Patent application title: Ii-Key/antigenic epitope hybrid peptide vaccines
Inventors:
Robert E. Humphreys (Acton, MA, US)
Minzhen Xu (Northborough, MA, US)
Minzhen Xu (Northborough, MA, US)
IPC8 Class: AC07K1400FI
USPC Class:
4241851
Class name: Drug, bio-affecting and body treating compositions antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same
Publication date: 2008-12-11
Patent application number: 20080305122
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Patent application title: Ii-Key/antigenic epitope hybrid peptide vaccines
Inventors:
Robert E. Humphreys
Minzhen Xu
Agents:
Kevin M. Farrell;Pierce Atwood
Assignees:
Origin: PORTSMOUTH, NH US
IPC8 Class: AC07K1400FI
USPC Class:
4241851
Abstract:
Disclosed is a nucleic acid molecule comprising a first expressible
sequence encoding a protein of interest or polypeptide of interest which
contains an MHC Class II-presented epitope. In addition, the nucleic acid
molecule comprises a second expressible nucleic acid sequence encoding an
antigen presentation enhancing hybrid polypeptide. The antigen
presentation enhancing hybrid polypeptide includes the following
elements: i) an N-terminal element consisting essentially of 4-16
residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1)
and non-N-terminal deletion modifications thereof that retain antigen
presentation enhancing activity; ii) a C-terminal element comprising an
MHC Class II-presented epitope in the form of a polypeptide or
peptidomimetic structure which binds to the antigenic peptide binding
site of an MHC class II molecule, the MHC Class II-presented epitope
being contained in the protein of interest of step a); and iii) an
intervening peptidyl structure linking the N-terminal and C-terminal
elements of the hybrid, the peptidyl structure having a length of about
20 amino acids or less.Claims:
1. A non-naturally occurring protein or polypeptide modified by
recombinant DNA techniques, comprising:a) a C-terminal element comprising
an MHC Class II-presented epitope;b) an N-terminal element comprising an
Ii-key motif; andc) an intervening element comprising a sequence from
about 4 to about 11 amino acid residues;the modification by recombinant
DNA techniques taking place within elements b) and c).
2. The protein or polypeptide of claim 1 wherein the Ii-Key motif comprises a segment of 5 contiguous amino acid residues containing at least two amino acids selected from the group consisting of LIVFM (SEQ ID NO: 790) and at least one selected from the group consisting of HKR.
3. The method of claim 1 wherein the MHC Class II-presented epitope is a molecular feature in a therapeutically significant protein.
4. An expressible nucleic acid sequence encoding a non-naturally occurring protein or polypeptide, the protein or polypeptide comprising:a) a C-terminal element comprising an MHC Class II-presented epitope;b) an N-terminal element comprising an Ii-key motif, the Ii-Key motif comprising a segment of 5 contiguous amino acid residues containing at least two amino acids selected from the group consisting of LIVFM (SEQ ID NO: 790) and at least one selected from the group consisting of HKR; andc) an intervening element comprising a sequence from about 4 to about 11 amino acid residues.
5. A method for suppressing an immune response directed toward an MHC Class II-presented epitope of interest, the method comprising:a) providing a nucleic acid sequence encoding the MHC Class II-presented epitope of interest, the nucleic acid sequence encoding an Ii-Key motif located 4-11 amino acids upstream from the N-terminal residue of the MHC Class II-presented epitope of interest; andb) modifying the Ii-Key motif to decrease its conformance to the archetypal Ii-Key regulatory motif, the archetype Ii-Key regulatory motif comprising a segment of 5 contiguous amino acids comprising at least two amino acids selected from the group consisting of leucine, isoleucine, valine, phenylalanine and methionine, and at lease one amino acid selected from the group consisting of histidine, lysine and arginine.
6. A method for enhancing an immune response directed toward an MHC Class II-presented epitope of interest, the method comprising:a) providing a nucleic acid sequence encoding the MHC Class II-presented epitope of interest, the nucleic acid sequence lacking an Ii-key motif located 4-11 amino acids upstream from the N-terminal residue of the MHC Class II-presented epitope of interest; andb) modifying the nucleic acid sequence to introduce an Ii-key motif appropriately spaced from the MHC Class II-presented epitope.
7. A method for immunizing a mammal, thereby altering immunological sensitivity toward a predetermined epitope or determinant, the method comprising:a) providing a first nucleic acid sequence comprising a first expressible sequence encoding a protein of interest, the protein of interest corresponding to a protein encoded by an infectious pathogen;b) providing a second nucleic acid sequence comprising a second expressible sequence encoding an antigen presentation enhancing hybrid polypeptide comprising:i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity;ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being contained in the protein of interest of step a); andiii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less; andc) administering the nucleic acid sequence of step b) to a patient in an amount sufficient to stimulate an immune response; andd) following T cell expansion in response to the administration of step c), administering the first nucleic acid sequence of step a) to the patient in an amount sufficient to stimulate an immune response.
8. A method for immunizing a mammal, thereby altering immunological sensitivity toward a predetermined epitope or determinant, the method comprising:a) providing the genome of a vaccine pathogen of interest;b) incorporating into the genome of step a) an expressible nucleic acid sequence encoding an antigen presentation enhancing hybrid polypeptide comprising:i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1 and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity;ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being encoded by the genome of step a); andiii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less;c) administering the nucleic acid sequence of step b) to a patient in an amount sufficient to stimulate an immune response; andd) following T cell expansion in response to the administration of step c), administering the first nucleic acid sequence of step a) to the patient in an amount sufficient to stimulate an immune response.
9. A method for displaying a virus-specific antigenic epitope of interest on the surface of an MHC Class II molecule-positive cell in which Ii protein expression is suppressed, the method comprising:a) providing an MHC Class II-positive cell; andb) introducing into the cell of step a) a recombinant virus comprising:i) an expressible nucleic acid sequence encoding the virus-specific antigenic epitope of interest; andii) an expressible reverse gene construct which encodes an RNA molecule which is complementary to an mRNA molecule which encodes human Ii protein, the RNA molecule having the ability to hybridize with the mRNA molecule thereby inhibiting translation of the mRNA molecule.
10. A method of claim 9 wherein the recombinant virus is a vaccinia virus.
Description:
BACKGROUND OF THE INVENTION
[0001]The immune system responds to foreign pathogens, to tumor cells, to autoimmune disease-inducing processes, to allergens, to grafts, through the recognition of the `foreign` or `abnormal` structures, as antigens. Most of those antigens are proteins, which are synthesized either by cells of the host, or by a pathogen. Such antigens are processed (proteolytically digested) into peptide fragments which come to be presented to the responding lymphocytes of the immune system, in a peptide-presenting structure on the surface of the antigen presenting cell. Those peptide presenting structures are called major histocompatibility complex (MHC) molecules. They obtained that name since they were first recognized as products of polymorphic, allelic genes in the MHC locus, which genes control graft rejection among inbred strains of mice.
[0002]The immune response to a specific antigen is mediated by T lymphocytes which recognize peptide fragments of those antigens in the MHC molecules. Within an antigen presenting cell (APC), peptide fragments of a proteolytically processed antigen become bound into the antigenic peptide binding site of major histocompatibility complex (MHC) molecules. These peptide-MHC complexes are then transported to the cell surface for recognition (of both the foreign peptide and the adjacent surface of the presenting MHC molecule) by T cell receptors on responding T lymphocytes. Those T lymphocytes can have either immunoregulatory functions (to help or suppress an immune response) or effector functions (to clear the pathogen or tumor, for example, through a cytotoxic immune response). The antigen-specific recognition event initiates the immune response cascade which leads to a protective immune response, or in the case of autoimmune processes, a deleterious immune response.
[0003]Two classes of MHC molecules function as immune system presenters of antigenic peptides to T cells. MHC class I molecules receive peptides from endogenously synthesized proteins, such as an infectious virus, in the endoplasmic reticulum about the time of synthesis of the MHC class I molecules. The MHC class I-bound antigenic peptides are presented at the cell surface to CD8-positive cytotoxic T lymphocytes, which then become activated and can directly kill the virus-expressing cells. In contrast, MHC class II molecules are synthesized in the endoplasmic reticulum with their antigenic peptide binding sites blocked by the invariant chain protein (Ii). These complexes of MHC class II molecules and Ii protein are transported from the endoplasmic reticulum to a post-Golgi compartment where Ii is released by proteolysis and a specific antigenic peptide becomes bound to the MHC class II molecule (Blum et al., Proc. Natl. Acad. Sci. USA 85: 3975 (1988); Riberdy et al., Nature 360: 474 (1992); Daibata et al., Mol. Immunol. 31: 255 (1994); Xu et al., Mol. Immunol. 31: 723 (1994); Xu et al., Antigen Processing and Presentation, Academic Press, NY p 227 (1994); Kropshofer et al., Science 270: 1357 (1995); and Urban et al., J. Exp. Med. 180: 751 (1994)).
[0004]R. Humphreys (1996) U.S. Pat. No. 5,559,028, and Humphreys et al. (1999) U.S. Pat. No. 5,919,639 revealed the mechanisms by which Ii protein is cleaved, releasing fragments in the course of cleavage to regulate the binding and locking in of antigenic peptides within the antigenic peptide binding site of MHC class II molecules (Adams et al., Eur. J. Immunol. 25: 1693 (1995); Adams et al., Arzneim. Forsch./Drug Research 47: 1069 (1997); and Xu et al., Arzneim. Forsch./Drug Research in press (1999)). One segment of the Ii protein, Ii(77-92), was found to act at an allosteric site outside the antigenic peptide binding site near the end of that site holding the N-terminus of the antigenic peptide. The referenced patents, furthermore, disclosed novel therapeutic compounds and methods to control this initial regulatory, antigenic peptide recognizing event of the immune response by three classes of mechanisms. In the first mechanism, antigenic peptides are spilled from cell surface MHC class II molecules by the action of compounds of the invention.
[0005]In the second, the charging of the antigenic peptide binding site on those molecules is promoted with compounds of the invention for binding of other, synthetic peptides. Such inserted peptide sequences can be either antigenic epitopes or nonantigenic peptide sequences which nevertheless bind tightly to block the antigenic peptide binding site. The third mechanism involves altering the rates of association/dissociation of antigenic peptides from those complexes and the nature of the interaction of components of the trimolecular MHC molecule/antigenic peptide/T cell receptor complex, and furthermore the interaction of that trimolecular complex with auxiliary cell-to-cell interaction molecules, in a manner to regulate differentiation and function of the responding T lymphocytes.
[0006]The identification of the mechanisms referred to above opens new avenues of therapeutic intervention. New methods and compositions based on these discoveries offer the promise of epitope-specific therapies.
SUMMARY OF THE INVENTION
[0007]In one aspect, the present invention relates to a nucleic acid molecule comprising a first expressible sequence encoding a protein of interest or polypeptide of interest which contains an MHC Class II-presented epitope. In addition, the nucleic acid molecule comprises a second expressible nucleic acid sequence encoding an antigen presentation enhancing hybrid polypeptide. The antigen presentation enhancing hybrid polypeptide includes the following elements: i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity; ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being contained in the protein of interest of step a); and iii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less.
[0008]In preferred embodiments, the modifications of the I-Key peptide include deletion of amino acids from the C-terminus; N-terminal extensions; and amino acid substitutions. In other embodiments, the C-terminal element further comprises an MHC Class I-presented epitope, or a portion thereof, the amino acid residues comprising the MHC Class I-presented epitope or portion thereof being constituent residues of the MHC Class II-presented epitope.
[0009]Disclosed embodiments also include embodiments wherein the C-terminal element further comprises an antibody-recognized determinant, or a portion thereof. The amino acid residues comprising the antibody-recognized determinant, or a portion thereof, are preferably constituent residues of the MHC Class II-presented epitope. Embodiments are disclosed in which the infectious pathogen is selected from the group consisting of anthrax, EBOLA, HIV and influenza. In addition to the disclosed compositions, methods of use are also described.
DETAILED DESCRIPTION OF THE INVENTION
[0010]As discussed in the Background of the Invention section of the present disclosure, U.S. application Ser. No. 09/396,813 (now U.S. Pat. No. 6,432,409) discloses hybrid peptides useful in connection with modulation of the immune system (referred to herein as "the '813 enhancing hybrid peptide"). The disclosure was based on the discovery that an MHC Class II-restricted antigenic epitope which is covalently linked to a mammalian Ii key peptide by an appropriate intervening chemical structure, to form a hybrid polypeptide, is presented to T lymphocytes by antigen presenting cells with significantly higher efficacy than is the precursor antigenic epitope. The disclosure of U.S. Pat. No. 6,432,409 is incorporated herein by reference.
[0011]The hybrid polypeptide disclosed was referred to as an "MHC Class II antigen presentation enhancing hybrid polypeptide", or more simply as an "enhancing hybrid". In this disclosure, such peptides have also been referred to as "Ii-Key/antigenic epitope hybrids" or "hybrid peptides". Alternatively, short-hand designations based on functional elements may be used, particularly in the Exemplification section. For example, Ii-Key/MHC Class II-presented antigenic epitope hybrids, Ii-Key/MHC Class II-presented antigenic epitope/MHC Class I-presented antigenic epitope hybrids, Ii-Key/MHC Class II-presented antigenic epitope/antibody-recognized determinant (ARD) hybrids. The preceding listing of alternative terminology may not be comprehensive, but reference to such enhancing hybrids will be clear in context.
[0012]The '813 enhancing hybrid has an N-terminus comprised of a mammalian Ii-Key peptide, or a modification thereof, which retains antigen presentation enhancing activity. Covalently, but indirectly, linked to the Ii-Key peptide is the specific MHC Class II antigenic epitope to be presented. Between the Ii-Key peptide and the antigenic epitope is an intervening chemical structure which covalently links the other two components. This intervening chemical structure was referred to simply as a "spacer". Necessary parameters of the spacer were described in detail.
[0013]The present disclosure specifically contemplates enhancing hybrid peptides containing antigenic epitopes/determinants in addition to the MHC Class II antigenic epitope disclosed in connection with the '813 enhancing hybrid. For example, the enhancing hybrids of the present invention may contain multiple MHC Class II epitopes. The inclusion of multiple MHC Class II epitopes enables a greater fraction of the human population to be immunized because the multiple epitopes are frequently presented by different alleles. In addition to a plurality of MHC Class II epitopes, the present invention also contemplates the inclusion of one or more MHC Class I epitopes and/or one or more ARDs (Antibody Recognized Determinants). The expressions "epitopes" and "determinants" are considered as synonyms by many skilled in the art. The use of the expression "epitope/determinant", as used herein, is intended to encompass MHC Class II epitopes, MHC Class I epitopes and ARDs.
[0014]The Exemplification section which follows provides numerous specific examples of experimentally-determined or predicted MHC Class II epitopes, MHC Class I epitopes and ARDs, which can be incorporated in enhancing hybrid peptides. The experimentally determined epitopes are preferred over algorithm-predicted epitopes for preclinical trials in animal models for human disease, in part, because a significant percentage of algorithm-predicted epitopes are not found to be biologically functional. Nevertheless, the "significant percentage" is sufficiently small that such epitopes are a source of sequences for the development of enhancing hybrids. In the context of a focus on a particular disease or condition, reference is made to the compounds and methods of use described in the corresponding Exemplification section which follows.
[0015]As will be discussed below, the use of the '813 enhancing hybrid peptide to enhance or augment an MHC Class II-mediated immune response, created an untapped immune reservoir. As will be discussed in greater detail below, the interaction of the '813 enhancing peptide with cells of the immune system greatly amplified a number of responsive cell types. Molecular input for a subset of these responsive cell types, in the form of the MHC Class II epitope component of the enhancing hybrid, were provided. However, large numbers of primed and responsive immune cell types were stimulated by the '813 peptide, but no provision for appropriate molecular inputs was provided. Such additional molecule inputs, in the form of MHC Class I epitopes and ARDS, is provided herein.
[0016]More specifically, the enhancement of the T helper cell stimulation mediated by the Class II epitope of the '813 peptide is substantially augmented (i.e., about 250 times) by the effect of the Ii-Key moiety. The clonal expansion of an immunoregulatory cell type, such as an activated T cell, has a cascading effect through the immune system. As discussed above, this can create an excess of immune capacity which has not been addressed in the prior art.
[0017]Ultimately, an MHC Class II-presented antigen which is an element of the hybrid peptide (either an enhancing hybrid peptide of the present invention or an '813 enhancing hybrid peptide), exerts its influence through presentation by an MHC Class II molecule on the surface of an antigen presenting cell. Two particularly important classes of antigen presenting cells are dendritic cells and macrophages. These antigen presenting cells have on their respective surfaces two types of special molecules that function in antigen presentation. These two types of molecules are MHC Class I and MHC Class II molecules. Antigenic peptides (e.g., MHC Class I or MHC Class II epitopes) are noncovalently bound to MHC Class I or MHC Class II molecules for subsequent presentation to antigen-specific receptors on T cells.
[0018]While not wishing to be bound by theory, it is thought that peptides containing MHC Class I and/or MHC Class II epitopes may be displayed on the surface of an antigen presenting cell in association with the cognate display molecule (i.e., MHC Class I molecules or MHC Class II molecules) through at least two mechanisms. For example, following contact with an antigen presenting cell, such peptides may be internalized by the antigen presenting cell and processed through classical channels. Alternatively, the MHC Class I or MHC Class II-presented antigen portion of such a peptide may bind directly to an MHC Class I or MHC Class II molecule on the surface of an antigen presenting cell. Thus, in both cases, the MHC Class I or MHC Class II-presented epitope of the peptide is displayed on the surface of an antigen presenting cell in association with its cognate MHC Class I or MHC Class II molecule.
[0019]Such an MHC Class II-associated display triggers a cascade of immune-mediated effects including the induction of T cells and the subsequent expansion of this induced population. T helper cells, stimulated in this manner, respond in a variety of ways. For example, stimulated T helper cells function by releasing cytokines that provide various activation signals for B cells. B cells produce a surface immunoglobulin which can recognize and specifically bind to an ARD element which is present, for example, on a protein or peptide which contacts the cell surface. The protein or peptide is then internalized and any processed MHC Class I or MHC Class II-presented epitopes present are subsequently displayed on the B cell surface in association with MHC Class I or MHC Class II molecules, respectively.
[0020]The example of an ARD-containing molecule provided in the preceding paragraph was a protein or peptide. In connection with the present invention, the ARD is provided as an element of an enhancing hybrid peptide. As was the case in the previous example, the enhancing hybrid peptide is internalized by the B cell and any MHC Class II epitopes present as an element of the enhancing hybrid are processed for display on the surface of the B cell in association with MHC Class II molecules. Such presentation further stimulates the helper T cell population resulting in proliferation and maturation of B lymphocytes to plasma cells which produce the antibody specific to the ARD.
[0021]The enhancing hybrid polypeptide of the present invention is comprised of 3 elements, as was the '813 enhancing hybrid. The 3 elements are: 1) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1 and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity; 2) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC Class II molecule; and 3) an intervening chemical structure covalently linking the N-terminal and C-terminal elements of the hybrid, the chemical structure being a covalently joined group of atoms which when arranged in a linear fashion forms a flexible chain which extends up to the length of 20 amino acids likewise arranged in a linear fashion.
[0022]The included additional epitope(s) or determinant(s) which distinguish the enhancing hybrid of the present invention from the '813 enhancing hybrid are preferably located within the C-terminal element or the linker element. Additionally, an epitope or determinant may overlap the C-terminal element and the linker element. In some circumstances it may be possible for an additional epitope or determinant to overlap between the linker element and the N-terminal Ii-Key moiety.
[0023]Generally speaking, MHC Class I and MHC Class II epitopes are comprised of from about 8 to about 12 amino acid residues. ARD elements are typically have a size range somewhat broader than MHC Class I and MHC Class II epitopes. A commonly cited size range for ARDs is from about 6 to about 16 amino acid residues. ARDs are recognized based on their 3-dimensional structure whereas MHC Class I and MHC Class II epitopes are recognized on the basis of their linear, primary amino acid structure.
[0024]To provide specificity to the options outlined in the preceding paragraph, it is necessary to discuss the anatomy of the enhancing peptide of the present invention in greater detail. The linker sequence has been described as an intervening chemical structure covalently linking the N-terminal and C-terminal elements of the hybrid, the chemical structure being a covalently joined group of atoms which when arranged in a linear fashion forms a flexible chain which extends up to the length of 20 amino acids likewise arranged in a linear fashion. Thus, to the extent that the linker sequence is comprised of amino acids (which is not a requirement), the disclosure of the present invention provides an additional functionality to the amino acid residues of the linker, above and beyond their required role as space occupiers.
[0025]The specified linker length (up to 20 amino acids arranged in a linear fashion) is long enough to contain a second complete MHC Class II epitope, a first complete MHC Class I epitope, or a first complete ARD or segments of such additional epitopes. Additionally, such a sequence length can accommodate a plurality of non-overlapping epitopes selected from the group consisting of MHC Class I epitopes, MHC Class II epitopes and ARDs.
[0026]It is known in the art that functional MHC Class I epitopes, MHC Class II epitopes and ARDs may be arranged in an overlapping manner while retaining full functionality of all represented epitopes. The respective functions of each epitope within a hybrid are not co-expressed at one point in time on a per peptide basis, because such peptides must be bound into MHC Class I or MHC Class II molecules and recognized in a folded structure by an antibody. Nevertheless, given a population of injected peptides with respective processing and/or binding to cell surface MHC molecules, all three classes of epitopes within any one Ii-Key enhancing hybrid can be effective immunogens within an immunized animal.
[0027]Minimum sequences are preferred for several reasons. These include simplicity and cost of synthesis, less opportunity for proteolytic degradation, less opportunity for metabolic change leading to clearance or adsorption. Thus, the linker element may contain a plurality of epitopes which overlap one another (i.e., an individual amino acid residues may be a components of more than one epitope). Similarly, the C-terminal element which includes an MHC Class II-presented epitope may also contain additional epitopes (MHC Class I, MHC Class II or ARD) in an overlapping or non-overlapping arrangement.
[0028]It is noted that the boundaries between the various elements of the enhancing hybrid peptide of the present invention are, within certain stated limits, somewhat arbitrary. Epitopes spanning the junctions between the various elements are encompassed within the scope of the present invention. Thus, for example, where a claim specifies that a portion of an epitope is contained within one of the enhancing hybrid peptide elements or domains (e.g., the linker region), this necessarily implies that the remaining portion is found in a contiguous portion of a flanking portion or domain. Partial (i.e., non-functional epitopes are of no utility in connection with the present invention).
[0029]Early work in this area demonstrated that the mammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1), and a modified mammalian Ii-key peptide, YRMKLPKPPKPVSKMR (SEQ ID NO: 2), have the ability to alter presentation of certain MHC Class II-restricted, antigenic peptides to T lymphocyte-hybridomas which recognize those respective antigenic peptides (U.S. Pat. No. 5,559,028; U.S. Pat. No. 5,919,639, the disclosures of which are incorporated herein by reference). Previous experimentation with modified versions of the Ii-key peptide have indicated that a wide variety of modifications can be made to this polypeptide without detriment to activity. Indeed, modifications often enhanced antigen presentation activity of the polypeptide.
[0030]Results detailed in the Exemplification section of U.S. application Ser. No. 09/396,813, now U.S. Pat. No. 6,432,409, indicate that all modified Ii key peptides which retain antigen presentation enhancing activity will function in the enhancing hybrid of the present invention when appropriately incorporated. Modifications of the Ii key peptide include deletion of one or more amino acids from the C-terminus, protection of the N-terminus, amino acid substitutions, and introduction of cyclical peptides. Deletions of the Ii key peptide which retain at least 4 contiguous amino acids of the original sequence, or a substituted version thereof, exhibit functional activity. Various natural or non-natural amino acids may be substituted at respective residue positions. Some examples of molecules which may be substituted are peptidomimetic structures, D-isomer amino acids, N-methyl amino acids, L-isomer amino acids, modified L-isomer amino acids, and cyclized derivatives. In addition, procedures of medicinal chemistry may be applied by one skilled in the art using routine experimental methods to obtain additional modifications of the N-terminal segment of hybrids. Examples of such procedures are methods of rational drug design, molecular modeling based on structural information from X-ray diffraction data, nuclear magnetic resonance data, and other computational methods, and screening of products of combinatorial chemical syntheses, and isolations of natural products. Examples of modified versions of Ii key peptide which are known to retain high activity are LRMK (SEQ ID NO: 3), LRMKLPK (SEQ ID NO: 4), LRMKLPKS (SEQ ID NO: 5), LRMKLPKSAKP (SEQ ID NO: 6), and LRMKLPKSAKPVSK (SEQ ID NO: 7). Other modifications and modified versions of the Ii-key peptide are described in U.S. Pat. No. 5,919,639, and U.S. Pat. No. 5,559,028. A modified version of the Ii-key peptide (YRMKLPKPPKPVSKMR, SEQ ID NO: 2) which is known to retain activity is referred to herein as an `Ii-key homolog`. The term Ii key homolog as used herein is inclusive of the Ii key peptide itself.
[0031]Such Ii-Key peptides were demonstrated by several experimental methods to bind to an allosteric site at the end of the antigenic peptide binding site of MHC Class II molecules holding the N-terminal end of an antigenic peptide. That process of binding to the allosteric site, facilitated the release and exchange of endogenously bound antigenic peptide with cell surface MHC Class II molecules.
[0032]Peptide homologs of the Ii-Key peptide act on murine or human MHC Class II molecules to promote the release of bound antigenic peptides and their replacement with synthetic peptides (Adams S. Arneimittelforschung. 1997 47:1069-1077; Xu M. Arneimittelforschung. 1999 49:791-9). Hybrid constructs of the Ii-Key peptide linked to an antigenic epitope peptide through either a simple polymethylene linker or the extended, natural sequence of the Ii protein, have 500 to 2000 times the potency of presentation versus the antigenic peptides (Humphreys R E. Vaccine. 2000 18:2693-2697). This property has great clinical utility in diagnosis, treatment monitoring and therapy of various diseases and conditions, as presented herein. This activity of the Ii-Key moiety within Ii-Key/antigenic epitope hybrids is found either in vitro or in vivo. This activity can be ascribed to interaction with cell surface MHC Class II molecules because the Ii-Key compounds were active in vitro with either living or paraformaldehyde-fixed antigen presenting cells (Adams S. Eur J Immunol. 1995 25:1693-1702). However, since the compounds are potent in vivo, they may also be taken up by the pathway which processes exogenous antigens and bind to MHC Class II molecule sin the post-Golgi, antigen charging compartment.
[0033]The MHC Class I epitopes, MHC Class II epitopes and ARDs of the enhancing hybrid of the present invention have been discussed above. Such epitopes/determinants selected for use in the generation of an enhancing hybrid of the present invention may be further modified for use. That is to say, polypeptides of natural or modified sequence, peptidomimetic structures, and also chemical structures which are not natural or modified amino acids may be included in the epitope/determinant elements of the enhancing hybrids disclosed herein. In addition, various chemical modifications may be made to the antigenic epitope/determinant element of the enhancing hybrid. For example, the addition, in whole or in part, of non-natural amino acids, or of other backbone or side chain moieties, wherein the modifications preserve binding specificities of the antigenic epitope/determinant. Such chemical structures might bear moderate, little, or no apparent structural resemblance to any antigenic peptide which is derived from a natural protein sequence. Such modifications might or might not bear on recognition by T cell receptors. Modifications may increase recognition of the antigenic epitope (e.g. lead to recognition by previously non-recognizing subsets of T cell receptors).
[0034]The intervening chemical structure, or spacer, has been discussed above. Where the intervening chemical structure comprises one or more epitopes/determinants, the overall length within defined limits is dictated to a large extent by the identity and of the epitope/determinant. In the case in which the intervening chemical structure is antigenically neutral, the teachings of U.S. application Ser. No. 09/396,813, now U.S. Pat. No. 6,432,409, apply. As indicated, the spacer is preferably less than the length of a peptidyl backbone of 9 amino acids linearly arranged. Optimally, spacer length is the length of a peptidyl backbone of between 4 and 6 amino acids, linearly arranged. Preferably, the spacer is unable to hydrogen bond in any spatially distinct manner to other distinct elements of the enhancing hybrid peptide.
[0035]Again, with respect to antigenically neutral spacer elements, various chemical groups may be incorporated in the spacer segment instead of amino acids. Examples are described in U.S. Pat. No. 5,910,300, the contents of which are incorporated herein by reference. In a preferred embodiment the spacer is comprised of an aliphatic chain optimally interrupted by heteroatoms, for example a C2-C6 alkylene, or ═N--(CH2)2-6--N═. Alternatively, a spacer may be composed of alternating units, for example of hydrophobic, lipophilic, aliphatic and aryl-aliphatic sequences, optionally interrupted by heteroatoms such as O, N, or S. Such components of a spacer are preferably chosen from the following classes of compounds: sterols, alkyl alcohols, polyglycerides with varying alkyl functions, alkyl-phenols, alkyl-amines, amides, hydroxyphobic polyoxyalkylenes, and the like. Other examples are hydrophobic polyanhydrides, polyorthoesters, polyphosphazenes, polyhydroxy acids, polycaprolactones, polylactic, polyglycolic polyhydroxy-butyric acids. A spacer may also contain repeating short aliphatic chains, such as polypropylene, isopropylene, butylene, isobutylene, pentamethlyene, and the like, separated by oxygen atoms.
[0036]Additional peptidyl sequences which can be used in a spacer are described in U.S. Pat. No. 5,856,456, the contents of which are incorporated herein by reference. In one embodiment, the spacer has a chemical group incorporated within which is subject to cleavage. Without limitation, such a chemical group may be designed for cleavage catalyzed by a protease, by a chemical group, or by a catalytic monoclonal antibody. In the case of a protease-sensitive chemical group, tryptic targets (two amino acids with cationic side chains), chymotryptic targets (with a hydrophobic side chain), and cathepsin sensitivity (B, D or S) are favored. The term `tryptic target` is used herein to describe sequences of amino acids which are recognized by trypsin and trypsin-like enzymes. The term `chymotryptic target` is used herein to describe sequences of amino acids which are recognized by chymotrypsin and chymotrypsin-like enzymes. In addition, chemical targets of catalytic monoclonal antibodies, and other chemically cleaved groups are well known to persons skilled in the art of peptide synthesis, enzymic catalysis, and organic chemistry in general, and can be designed into the hybrid structure and synthesized, using routine experimental methods.
[0037]Not all embodiments of the present invention include immunogenic neutrality of the intervening chemical structure, or spacer. That is, the present invention includes embodiments in which the intervening chemical structure, or spacer, is selected from the group consisting of: 1) an MHC Class I epitope, or a portion thereof; and 2) an antibody-recognized determinant, or a portion thereof. In particular, this embodiment is important in connection with the anticipated filing of a counterpart International Application for which the continuation-in-part provisions of the U.S. patent law are inapplicable.
[0038]The hybrids of the present invention vary from totally peptide in character to substantially non-peptide in character. In view of the fact that some homologs are substantially reduced or non-peptide in character, they will be more likely to have favorable properties, for example, penetration through cellular membranes, solubility, resistance to proteolysis, resistance to inactivation by conjugation, oral bioavailability, and longer half life in vivo.
[0039]Also included within the scope of this invention are pharmaceutically acceptable salts of the hybrid molecule, when an acidic or basic group is present in the structure. The term `pharmaceutically acceptable salt` is intended to include all acceptable salts such as acetate, ammonium salt, benzenesulfonate, benzoate, borate, bromide, calcium edetate, camsylate, carbonate, chloride/dihydrochloride, citrate, clavulanate, edetate, edisylate, estolate, esylate, fumarate, hexylresorcinate, hydrabamine, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamide, oleaste, oxalate, pamoate, palmitate, panoate, pantothenate, phosphate/diphosphate, polygalacturonate, subacetate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like. The pharmaceutically acceptable salt can be used as a dosage form for modifying the solubility or hydrolysis characteristics, or can be used in a sustained release or pro-drug formulation. Depending on the particular functionality for the compound of the present invention, pharmaceutically acceptable salts of the compounds of this invention may be formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and from bases such as ammonia, arginine, chloroprocaine, choline, diethanolamine, diethylamine, ethylenediamine, lysine, N-methyl-glutamine, ornithine, N,N'-dibenzylethylenediamine, N-benzylphenethylamine, piperazine, procaine, tris(hydroxymethyl)aminomethane, and tetramethylenediamine hydroxide, and the like. These salts may be prepared by standard procedures, for example, by reacting a free acid with suitable organic or inorganic base. When a basic group is present, such as an amino, and acidic salt, i.e., acetate, hydrobromide, hydrochloride, pamoate, and the like, can be used as the dosage form.
[0040]Also in the case of an acid (--COOH) or alcohol group being present, pharmaceutically acceptable esters can be employed, for example, acetate, maleate, pivaloyloxymethyl, and the like and those esters known in the art for modifying solubility or hydrolysis characteristics for use as sustained release or prodrug formulations.
[0041]The hybrid molecules of this present invention or components thereof may have chiral centers, and therefor may occur as racemates, racemic mixtures, and as individual enantiomers or diastereomers, with all such isomeric forms being included in the present invention as well as mixtures thereof. Furthermore, some of the crystalline forms of hybrid compounds of the present invention may exist as polymorphous and as such are intended to be included in the present invention. In addition, some of the compounds of the present invention may form solvates with water or common organic solvents. Such solvates are also encompassed within the scope of this invention.
[0042]The enhancing hybrid of the present invention may be composed of peptide or peptidomimetic or additional chemical groups which may be synthesized and selected by methods which have been developed for the synthesis and selection of antigenic peptides. Those methods and compounds are presented in the following patents: U.S. Pat. No. 4,708,871; U.S. Pat. No. 5,194,392; U.S. Pat. No. 5,270,170; U.S. Pat. No. 5,382,513; U.S. Pat. No. 5,539,084; U.S. Pat. No. 5,556,762; (1997) U.S. Pat. No. 5,595,915; U.S. Pat. No. 5,747,334; and U.S. Pat. No. 5,874,214, the contents of which are incorporated herein by reference.
[0043]The disclosure presented above relates primarily to antigen presentation enhancing hybrid peptides. In another aspect, the present invention relates to nucleic acid sequences which encode such enhancing peptides. It is noted that the scope of the enhancing hybrid peptide disclosure is somewhat broader than the corresponding nucleic acid sequence disclosure in light of the fact that enhancing hybrid peptides produced using recombinant DNA techniques from an encoding nucleic acid sequence must be produced from one of the 20 naturally occurring amino acids. A much broader range of substitutions is available when an enhancing hybrid peptide is produced by chemical synthetic techniques.
[0044]A wide variety of delivery systems are available for use in delivering the enhancing hybrid of the present invention to a target cell in vitro and in vivo. Such delivery systems include, for example, viral and non-viral systems. Examples of suitable viral systems include, for example, adenoviral vectors, adeno-associated virus, retroviral vectors, vaccinia, herpes simplex virus, HIV, the minute virus of mice, hepatitis B virus and influenza virus. Non-viral delivery systems may also be used, for example using, uncomplexed DNA, DNA-liposome complexes, DNA-protein complexes and DNA-coated gold particles, bacterial vectors such as salmonella, and other technologies such as those involving VP22 transport protein, Co-X-gene, and replicon vectors.
[0045]One option for expressing a nucleic acid sequence of interest in an animal cell is the adenovirus system. Adenovirus possesses a double-stranded DNA genome, and replicates independently of host cell division. Adenoviral vectors offer a variety of advantages relative to alternative methods for introducing expressible constructs into cells. For example, adenoviral vectors are capable of transducing a broad spectrum of human tissues and high levels of gene expression can be obtained in dividing and nondividing cells. Adenoviral vectors are characterized by a relatively short duration of transgene expression due to immune system clearance and dilutional loss during target cell division. Several routes of administration can be used including intravenous, intrabiliary, intraperitoneal, intravesicular, intracranial and intrathecal injection, and direct injection of a target organ or tissue. Thus, it is recognized in the art that targeting based on anatomical boundaries is achievable.
[0046]The adenoviral genome encodes about 15 proteins and infection involves a fiber protein which binds to a cell surface receptor. This receptor interaction results in internalization of the virus. Viral DNA enters the nucleus of the infected cell and transcription is initiated in the absence of cell division. Expression and replication is under control of the E1A and E1B genes (see Horwitz, M. S., In Virology, 2nd ed., 1990, pp. 1723-1740). Removal of E1 genes renders the virus replication-incompetent.
[0047]Adenoviral serotypes 2 and 5 have been extensively used for vector construction. Bett et al. (Proc. Nat. Acad. Sci. U.S.A., 1994, 91: 8802-8806) have used an adenoviral type 5 vector system with deletions of the E1 and E3 adenoviral genes. The 293 human embryonic kidney cell line has been engineered to express E1 proteins and can thus transcomplement the E1-deficient viral genome. The virus can be isolated from 293 cell media and purified by limiting dilution plaque assays (Graham, F. L. and Prevek, L. In Methods in Molecular Biology: Gene Transfer and Expression Protocols, Humana Press 1991, pp. 109-128). Recombinant virus can be grown in 293 cell line cultures and isolated by lysing infected cells and purification by cesium chloride density centrifugation. A problem associated with the 293 cells for manufacture of recombinant adenovirus is that due to additional flanking regions of the E1 genes, they may give rise to replication competent adenovirus (RCA) during the viral particle production. Although this material is only wild-type adenovirus, and is not replication competent recombinant virus, it can have significant effects on the eventual yield of the desired adenoviral material and lead to increased manufacturing costs, quality control issues for the production runs and acceptance of batches for clinical use. Alternative cell lines such as the PER.C6 which have more defined E1 gene integration than 293 cells (i.e. contain no flanking viral sequence) have been developed which do not allow the recombination events which produce RCA and thus have the potential to overcome above viral production issues.
[0048]Adeno-associated virus (AAV) (Kotin, R. M., Hum. Gene Ther., 1994, 5: 793-801) are single-stranded DNA, nonautonomous parvoviruses able to integrate into the genome of nondividing cells of a very broad host range. AAV has not been shown to be associated with human disease and does not elicit an immune response. AAV has two distinct life cycle phases. Wild-type virus will infect a host cell, integrate and remain latent. In the presence of adenovirus, the lytic phase of the virus is induced, which depends on the expression of early adenoviral genes, and leads to active virus replication. The AAV genome is composed of two open reading frames (called rep and cap) flanked by inverted terminal repeat (ITR) sequences. The rep region encodes four proteins which mediate AAV replication, viral DNA transcription, and endonuclease functions used in host genome integration. The rep genes are the only AAV sequences required for viral replication. The cap sequence encodes structural proteins that form the viral capsid. The ITRs contain the viral origins of replication, provide encapsidation signals, and participate in viral DNA integration. Recombinant, replication-defective viruses that have been developed for gene therapy lack rep and cap sequences. Replication-defective AAV can be produced by co-transfecting the separated elements necessary for AAV replication into a permissive 293 cell line. U.S. Pat. No. 4,797,368 contains relevant disclosure and such disclosure is incorporated herein by reference.
[0049]Retroviral vectors are useful for infecting dividing cells, and are composed of an RNA genome that is packaged in an envelope derived from host cell membrane and viral proteins. Retroviral gene expression involves a reverse transcription step in which its positive-strand RNA genome is employed as a template to direct the synthesis of double-stranded DNA, which is then integrated into the host cell DNA. The integrated provirus is able to use host cell machinery for gene expression.
[0050]Murine leukemia virus is a commonly employed retrovirus species (Miller et al., Methods Enzymol., 1993, 217: 581-599). Retroviral vectors are typically constructed by deletion of the gag, pol and env genes. The deletion of these sequences provides capacity for insertion of nucleic acid sequences of interest, and eliminates the replicative functions of the virus. Genes encoding antibiotic resistance often are included as a means of selection. Promoter and enhancer functions also may be included, for example, to provide for tissue-specific expression following in vivo administration. Promoter and enhancer functions contained in long terminal repeats may also be used.
[0051]Such viruses, and modifications of such viruses which carry an exogenous nucleic acid sequence of interest, can only be produced in viral packaging cell lines. The packaging cell line may be constructed by stably inserting the deleted viral genes (gag, pol and env) into the cell such that they reside on different chromosomes to prevent recombination. The packaging cell line is used to construct a producer cell line that will generate replication-defective retrovirus containing the nucleic acid sequence of interest by inserting the recombinant proviral DNA. Plasmid DNA containing the long terminal repeat sequences flanking a small portion of the gag gene that contains the encapsidation sequence and the genes of interest is transfected into the packaging cell line using standard techniques for DNA transfer and uptake (electroporation, calcium precipitation, etc.). Variants of this approach have been employed to decrease the likelihood of production of replication-competent virus (Jolly, D., Cancer Gene Therapy, 1994, 1, 51-64). The host cell range of the virus is determined by the envelope gene (env) and substitution of env genes with different cell specificities can be employed. Incorporation of appropriate ligands into the envelope protein may also be used for targeting.
[0052]Administration of recombinant retroviral vectors may be accomplished by any suitable technique. Such techniques include, for example, ex vivo transduction of patients' cells, direct injection of virus into tissue, and by the administration of the retroviral producer cells. ex vivo approaches require the isolation and maintenance in tissue culture of the patient's cells. In this context, a high ratio of viral particles to target cells can be achieved and thus improve the transduction efficiency (see, e.g., U.S. Pat. No. 5,399,346, the disclosure of which is incorporated herein by reference). U.S. Pat. No. 4,650,764 contains disclosure relevant to the use of retroviral expression systems and the disclosure of this referenced patent is incorporated herein by reference.
[0053]In some cases direct introduction of virus in vivo is necessary or preferred. Retroviruses have been used to treat brain tumors wherein the ability of a retrovirus to infect only dividing cells (tumor cells) may be particularly advantageous. The administration of a retrovirus producer cell line directly into a brain tumor in a patient has also been proposed (see e.g., Oldfield et al., Hum. Gene Ther., 1993, 4: 39-69). Such a producer cell would survive within the brain tumor for a period of days, and would secrete retrovirus capable of transducing the surrounding brain tumor.
[0054]Pox virus-based systems for expression have been described (Moss, B. and Flexner, C., Annu. Rev. Immunol., 1987, 5: 305-324; Moss, B., In Virology, 1990, pp. 2079-2111). Vaccinia, for example, are large, enveloped DNA viruses that replicate in the cytoplasm of infected cells. Nondividing and dividing cells from many different tissues are infected, and gene expression from a nonintegrated genome is observed. Recombinant virus can be produced by inserting the transgene into a vaccinia-derived plasmid and transfecting this DNA into vaccinia-infected cells where homologous recombination leads to the virus production. A significant disadvantage is that it elicits a host immune response to the 150 to 200 virally encoded proteins making repeated administration problematic.
[0055]The herpes simplex virus is a large, double-stranded DNA virus that replicates in the nucleus of infected cells. This virus is adaptable for use in connection with exogenous nucleic acid sequences (see Kennedy, P. G. E. and Steiner, I., Q. J. Med., 1993, 86: 697-702). Advantages include a broad host cell range, infection of dividing and nondividing cells, and large sequences of foreign DNA can be inserted into the viral genome by homologous recombination. Disadvantages are the difficulty in rendering viral preparations free of replication-competent virus and a potent immune response. Deletion of the viral thymidine kinase gene renders the virus replication-defective in cells with low levels of thymidine kinase. Cells undergoing active cell division (e.g., tumor cells) possess sufficient thymidine kinase activity to allow replication.
[0056]A variety of other viruses, including HIV, the minute virus of mice, hepatitis B virus, and influenza virus, have been disclosed as vectors for gene transfer (see Jolly, D., Cancer Gene Therapy, 1994, 1: 51-64). Nonviral DNA delivery strategies are also applicable. These DNA delivery strategies relate to uncomplexed plasmid DNA, DNA-lipid complexes, DNA-liposome complexes, DNA-protein complexes, DNA-coated gold particles and DNA-coated polylactide coglycolide particles. Purified nucleic acid can be injected directly into tissues and results in transient gene expression for example in muscle tissue, particularly effective in regenerating muscle (Wolff et al., Science, 1990, 247: 1465-1468). Davis et al. (Hum. Gene Ther., 1993, 4: 733-740) has published on direct injection of DNA into mature muscle (skeletal muscle is generally preferred).
[0057]Plasmid DNA on gold particles can be "fired" into cells (e.g. epidermis or melanoma) using a gene-gun. DNA is coprecipitated onto the gold particle and then fired using an electric spark or pressurized gas as propellant (Fynan et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90: 11478-11482). Electroporation has also been used to enable transfer of DNA into solid tumors using electroporation probes employing multi-needle arrays and pulsed, rotating electric fields (Nishi et al., in Cancer Res., 1996, 56:1050-1055). High efficiency gene transfer to subcutaneous tumors has been claimed with significant cell transfection enhancement and better distribution characteristics over intra-tumoral injection procedures.
[0058]Lipid-mediated transfections are preferred for both in vitro and in vivo transfections (Horton et al., J. Immunology, 162:6378, 1999). Lipid-DNA complexes are formed by mixing DNA and lipid 1 to 5 minutes before injection, using commercially available lipids such as DMRIE-C reagent.
[0059]Liposomes work by surrounding hydrophilic molecules with hydrophobic molecules to facilitate cell entry. Liposomes are unilamellar or multilamellar spheres made from lipids. Lipid composition and manufacturing processes affect liposome structure. Other molecules can be incorporated into the lipid membranes. Liposomes can be anionic or cationic. Nicolau et al. (Proc. Natl. Acad. Sci. U.S.A., 1983, 80: 1068-1072) has published work relating to insulin expression from anionic liposomes injected into rats. Anionic liposomes mainly target the reticuloendothelial cells of the liver, unless otherwise targeted. Molecules can be incorporated into the surface of liposomes to alter their behavior, for example cell-selective delivery (Wu, G. Y. and Wu, C. H., J. Biol. Chem., 1987, 262: 4429-4432).
[0060]Felgner et al. (Proc. Nat. Acad. Sci. U.S.A., 1987, 84: 7413-7417) has published work relating to cationic liposomes, demonstrated their binding of nucleic acids by electrostatic interactions and shown cell entry. Intravenous injection of cationic liposomes leads to transgene expression in most organs on injection into the afferent blood supply to the organ. Cationic liposomes can be administered by aerosol to target lung epithelium (Brigham et al., Am. J. Med. Sci., 1989, 298: 278-281). In vivo studies with cationic liposome transgene delivery have been published (see, e.g., Nabel, G., Rev. Hum. Gene Ther., 1994, 5: 79-92; Hyde et al., Nature, 1993, 362: 250-255 and; Conary et al., J. Clin. Invest., 1994, 93: 1834-1840).
[0061]Microparticles are being studied as systems for delivery of DNA to phagocytic cells such approaches have been reported by Pangaea Pharmaceuticals. Such a DNA microencapsulation delivery system has been used to effect more efficient transduction of phagocytic cells, such as macrophages, which ingest the microspheres. The microspheres encapsulate plasmid DNA encoding potentially immunogenic peptides which, when expressed, lead to peptide display via MHC molecules on the cell surface which can stimulate immune response against such peptides and protein sequences which contain the same epitopes. This approach is presently aimed towards a potential role in anti-tumor and pathogen vaccine development but may have other possible gene therapy applications.
[0062]Natural viral coat proteins which are capable of homogeneous self-assembly into virus-like particles (VLPs) have also been used to package DNA for delivery. The major structural coat protein (VP1) of human polyoma virus can be expressed as a recombinant protein and is able to package plasmid DNA during self-assembly into a VLP. The resulting particles can be subsequently used to transduce various cell lines.
[0063]Improvements in DNA vectors have also been made and are likely applicable to many of the non-viral delivery systems. These include the use of supercoiled minicircles (which do not have bacterial origins of replication nor antibiotic resistance genes and thus are potentially safer as they exhibit a high level of biological containment), episomal expression vectors (replicating episomal expression systems where the plasmid amplifies within the nucleus but outside the chromosome and thus avoids genome integration events) and T7 systems (a strictly a cytoplasmic expression vector in which the vector itself expresses phage T7 RNA polymerase and the therapeutic gene is driven from a second T7 promoter, using the polymerase generated by the first promoter). Other, more general improvements to DNA vector technology include use of cis-acting elements to effect high levels of expression, sequences derived from alphoid repeat DNA to supply once-per-cell-cycle replication and nuclear targeting sequences.
[0064]In other aspects, the present invention relates to methods for enhancing presentation of an MHC Class II-presented antigenic peptide to a T-lymphocyte. As discussed in U.S. Pat. No. 6,432,409, the MHC Class II-restricted antigenic epitope is appropriately incorporated into the C-terminus of an enhancing hybrid of the present invention, described above. The produced enhancing hybrid is then contacted under physiological conditions to an MHC Class II expressing antigen presenting cell which is in contact with or is then contacted to a T cell which is responsive to the presentation of the antigenic epitope by an MHC Class II molecule of the antigen presenting cell. This method is suitable for use with all antigenic epitopes which conform to the above listed description of an antigenic epitope. Examples of methods to assay such enhancement in vitro are detailed in the Exemplification section below, and in U.S. patents listed in the present disclosure.
[0065]In one aspect, the subject invention relates to a method to improve the potency of peptide vaccines containing MHC Class II-presented epitopes of antigens of interest to activate CD4+ immunoregulatory T cells for therapeutic or diagnostic purposes. A wide range of diseases and conditions in humans will benefit from the application of the compounds and methods of this invention to activate CD4+ immunoregulatory T cells. Such CD4+ immunoregulatory T cells can either augment or suppress the immune response to antigens of clinical interest in cancer, infectious disease, allergy, autoimmunity, graft rejection, and other clinical processes.
[0066]Antigens of clinical interest in the treatment or modification of various diseases and conditions as presented herein, are recognized by the T cells of the immune system as small peptide fragments, which are presented by Major Histocompatibility Complex (MHC) molecules on the surfaces of antigen presenting cells. MHC Class I molecules present such antigenic peptides to CD8+ cytotoxic or killer T cells. Most cells of the body express cell surface MHC Class I-presented peptides which have been drawn from the repertoire of cellular proteins and bound into the MHC Class I molecules of those cells at the time of their synthesis in the endoplasmic reticulum (the "immunological survey of self"). After viral infection or malignant transformation, the CD8+, cytotoxic T cells recognize the novel or "foreign" endogenously derived peptides in the MHC Class I molecules and kill the presenting cells.
[0067]MHC Class II molecules present antigenic peptides to CD4+ T immunoregulatory cells, which regulate the immune response by augmenting or suppressing various effector mechanisms of that response. Such effector mechanisms include, for example, cytotoxic T cell killing of target cells, antibody production by B cells and plasma cells, and dendritic cell activation. Because they regulate directly or indirectly almost all mechanisms in the immune response, CD4+ T immunoregulatory cells have been called the conductors of the immune response orchestra. MHC Class II molecules are expressed on only a subset of the cells of the body, such as macrophages, dendritic cells, and B-cells that have specialized mechanisms to internalize and process antigens of the environment. At the time of synthesis in the endoplasmic reticulum, the antigenic peptide-binding site of MHC Class II molecules is filled with the Ii protein. After transport of that complex to a post-Golgi, antigen charging compartment, the Ii protein is removed by proteases with the concerted insertion of antigenic peptides from foreign proteins, which have been internalized and processed by the antigen processing cells (Cresswell P. Cell. 1996 84:505-7; Hudson A W. Exp Cell Res. 2002 272:1-7; Bryant P W. Adv Immunol. 2002 80:71-114). The Ii-Key segment of the Ii protein interacts with an allosteric site on the MHC Class II molecule to induce lability of the antigenic peptide binding site during release of the Ii protein and binding of a selected antigenic peptide. After dissociation/destruction of the Ii-Key segment, the antigenic peptide is tightly bound in the MHC Class II molecule, for extended expression in the antigenic peptide binding site of those molecules. After transport to the cell surface, such MHC Class II-antigen peptide complexes are recognized by specialized receptors on CD4+ T immunoregulatory cells. Activation of those cells regulates the immune response in various ways, which are considered later in terms of individual therapeutic objectives. In brief, subsets of CD4+ cells may be activated along Th1, Th2, or Th2 pathways, which are characterized by differential induction of cytokines and other genes. Those regulatory cells either induce or suppress immune responses in an antigen-specific manner. Furthermore, CD4+ T cells can be induced to be a long-lived population of memory T cells.
[0068]The allosteric site at which the Ii-Key segment of the Ii protein interacts is accessible to the environment in cell surface-expressed MHC Class II molecules. This fact is of considerable value clinically because Ii-Key/antigenic epitope hybrids peptides can be administered in a simple manner in a fluid phase, for example subcutaneously, intravenously, intrathecally, intraperitoneally, transmucosally and as an aerosol to the respiratory tract, and can contact the target MHC Class II molecules without traversing membranes or undergoing any special intracellular or metabolic processing or modification. Furthermore, the fact that the allosteric site of MHC Class II molecules is expressed on the surfaces of living, or even paraformaldehyde-fixed antigen presenting cells has facilitated in vitro studies of the mechanism of action of Ii-Key peptides and of Ii-Key/antigenic epitope hybrid peptides, as presented both herein and previously in U.S. Pat. No. 5,559,028 (1996) and U.S. Pat. No. 5,919,639 (1999).
[0069]In addition to the favored property of contacting cell surface-expressed with MHC Class II molecules after a simple fluid phase administration, the Ii-Key/antigenic epitope hybrid peptides can also be taken up in an antigen processing and presenting cell, such as a macrophage or dendritic cell, and contacted to MHC Class II molecules in the course of their transversing a post-Golgi, antigen charging compartment. Selective use of either these two, very different pathways for antigen to contact MHC Class II molecules is useful during the treatment of various diseases and conditions as described herein. For example, intravenous administration at a low concentrations over a long period of time, will favor epitope presentation in a manner yielding immunosuppression, which is favored for example in the case of peptide epitopes from antigens related to multiple sclerosis or rheumatoid arthritis. Or, on the other hand, in the case of augmenting the immune response to a subsequently administered DNA vaccine for an antigen relevant to therapy of either a cancer or an infectious disease, administration of an Ii-Key/antigenic epitope incorporating an epitope coded by the DNA vaccine with an adjuvant cytokine or other stimulant promotes development of a Th1-mediated response.
[0070]The method of enhancing presentation of an MHC Class II-restricted antigenic epitope to a T lymphocyte finds wide application in the diagnosis and therapy of diseases. T cell responses to diagnostic antigenic epitopes are often measured in the diagnosis of diseases, particularly with respect to etiological infectious agents. The use of enhancing hybrids of the present invention which have such diagnostic antigenic epitopes incorporated will increase substantially the sensitivity of these in vitro diagnostic assays. In the case of infectious diseases and cancer, antigenic epitopes which are identified as pathogen or cancer specific can be incorporated into an enhancing hybrid of the present invention and the hybrid then used to initiate a Th response to a pathogen or cancer specific MHC Class II-presented antigenic epitope. This response leads to activation and expansion of T helper cells which in turn activate or `license` dendritic cells, to prime an effective MHC Class I restricted cytotoxic T lymphocyte response toward the invading organism. In the case of autoimmune diseases, allergy, and graft rejection, specific antigenic epitopes which trigger the pathogenic immune response are identified and then incorporated into an enhancing hybrid of the present invention. The hybrid is then used to stimulate T cells in a manner leading to a Th2 response which will down regulate T cell responses. In this case, stimulation of a suppressor cell response is used to down regulate a pathogenic immune response. Methods for identifying enhancing hybrids which specifically stimulate a predetermined subset of T lymphocytes are described below. Additional methods and utilities of such hybrids in the therapy of disease are considered below.
[0071]In another aspect, the Ii-Key antigenic epitope hybrids increase the repertoire of MHC Class II alleles, and therefore the reaction of individuals in the vaccinated population who can be immunized with any given MHC Class II-presented epitope. Since the potency of an antigenic epitope presented within an Ii-Key/antigenic epitope hybrid is much larger than that of the same epitope presented as a peptide, mammals with low responder MHC Class II alleles for that given epitope may be stimulated to a level equivalent to mammals with high responder MHC Class II alleles. The development of immunoregulatory T cell clones recognizing that epitope will lead to enhanced subsequent presentation of the same epitope from an antigen of interest, for example of a malignant or virus-infected cell. This expansion of the repertoire of MHC Class II alleles promoting a therapeutic response to any one epitope, leads to a greater portion of the population being protected by immunizing with any given epitope. Thus, a "basket of peptides" vaccine, i.e., one containing peptides with various epitopes, is not needed. That is, without the use of the Ii-Key/antigenic epitope hybrid, a much larger number of individual antigenic epitope peptides must be used in a T helper peptide vaccine.
[0072]In another aspect, Ii-Key/antigenic epitope hybrids enhance responses to DNA vaccines. Vaccines containing the cDNA sequence for one or more antigens from either a pathogen or a tumor specific or tumor-associated antigen are being tested clinically. However, in many instances, high levels of protective antibodies, or long duration immunological memory, or maximal cytotoxic T cell responses, are not found. This lack of potency has been ascribed to weak helper T cell responses to such immunization. T helper cells can therefore be primed with Ii-Key/antigenic epitope hybrids to MHC Class II-presented epitopes in the cDNA vaccine in a suitable temporal schedule to maximize immunization with the cDNA vaccine.
[0073]In another aspect, addition of the Ii-Key-linker to each of a member of a library of peptides, overlapping through the sequence of an antigen of interest, increases the sensitivity of picking up MHC Class II epitopes. Given the increased potency of presentation of epitope in such hybrids, weakly antigenic epitopes, and epitopes with other limitations in inducing a particular pathway of biological response, for example those mediated by IgE, might be better recognized. Furthermore, in the case of combinatorial libraries of peptides synthesized with homology to a given experimental antigenic epitope, or a sequence only partially identified, for example by HLPC separation and tandem mass spectrography, the potency of peptides in such libraries can be enhanced by synthesizing the Ii-Key motif and linker at the N-terminus of such peptides. The fact that the synthesis of such peptides proceeds from the C- to the N-termini is favorable because either, sequentially ava, then K, then M, then R, then L (LRMK in reverse) (SEQ ID NO: 8) can be added, or Ac-LRMK-ava (SEQ ID NO: 9) can be added terminally as a unit.
[0074]In another aspect Ii-Key/antigenic epitope technology can be applied in the discovery, validation and use of cryptic antigenic epitopes. Cryptic antigenic epitopes have been defined empirically to be those epitopes, which are recognized upon immunization of a mammal with a peptide from an antigenic protein, but not upon immunization of a genetically identical mammal with the intact antigenic protein. In extensive experimental studies by Sercarz and colleagues, a procedure was established to discover most cryptic epitopes in a given antigenic protein, with respect to a given strain of mice. A library of peptides, for example each 15 amino acids in length with overlapping terminal segment of 6 amino acids, was created through the primary amino acid sequence of an antigen protein of interest, for example hen egg lysozyme. One mouse of a given strain was immunized with lysozyme and the proliferative response of splenic T cells to each of the peptides in the lysozyme library was tested. The epitopes in peptides stimulating the proliferative response were termed dominant epitopes. When additional mice of that strain were immunized with each of the respective peptides of the library of lysozyme peptides, all of the dominant epitopes were found to be immunogenic in isolated peptides, but additional epitopes were also discovered. These additional experiments were termed cryptic epitopes. Sercarz and colleagues demonstrated a series of mechanisms by which cryptic epitopes are not immunogenic when presented within the intact proteins. The clinical value of cryptic epitopes lies in part in the fact that a given individual is very unlikely to have previously recognized such epitope immunologically and therefore has not been tolerized to that epitope. Upon presentation of such cryptic epitopes within Ii-Key hybrids, therefore, a robust immune response can be developed, if the dose, route, schedule and adjuvants are designed toward that end. In the case of cancer, and even in the case of some infectious agents, tolerance can be developed to one or more epitopes, with the end result being that an effective immune response of the host is blocked. Cryptic epitope offer a novel repertoire of antigenic epitopes for such therapeutic purposes. Likewise such epitopes from allergens offer targets to develop therapeutic Th1 response while an IgE-promoting Th2 response had been developed toward dominant epitopes of the allergen. In such cases, Ii-Key/antigenic epitope hybrids containing dominant epitopes might exacerbate the pathological allergic responses.
[0075]In another aspect, the Ii-Key/antigenic epitope hybrids are favored in clinical diagnostic or therapeutic immunizations of patient for responses to epitopes in antigen of interest. That is, immunizing with Ii-Key/antigenic epitope hybrids as opposed to the epitope peptide, is favored because the dose required to obtain a clinically significant result is greatly reduced. Concomitantly the likelihood of a fatal anaphylactic response to the antigen, either in the case of an allergen, or otherwise, is reduced.
[0076]Additional assay systems can be used to measure the effect of incorporating an antigenic epitope other than a single MHC Class II epitope into an enhancing hybrid of the present invention. Assays with alternative readouts include, without limitation, measuring efficacy of immunoglobulin production from B cells, measuring efficacy of cytotoxic T cell generation, and the use of native T cells from animals which are outbred, inbred, congenic, transgenic for a T cell receptor or another biologically relevant molecule.
[0077]Methods for modulating the immune response of an individual finds application in the therapeutic treatment of an individual with a disease or condition. An antigenic epitope to which an enhanced immune response is considered to be beneficial in treatment of the patient is first selected. In one embodiment, the molecule from which the antigenic epitope is derived plays a role in pathogenesis. Alternatively, the antigenic epitope may be an epitope found on a harmful agent such as a pathogen, or on a pathogen infected cell. The term `therapeutic treatment` as used herein is intended to include ameliorating the signs or symptoms of disease, or arresting the progression of disease in an individual identified or considered to be suffering from a disease. The term `prevention` as used herein is intended to include ameliorating the underlying cause to, or associated factor predisposing to, a disease, in an individual who might not have begun to experience recognizable signs or symptoms of a disease.
[0078]The disease may be an infectious disease caused or associated with infection by a bacterium, a virus, a parasite, a fungus, a rickettsia, or other infectious agent, or combination of such agents. The therapy may be directed against the toxin of a disease or against a receptor for a toxin of a disease. Preferred toxins for epitope derivation include, without limitation, staphylococcal enterotoxins, toxic shock syndrome toxin, retroviral antigens (e.g. antigens derived from human immunodeficiency virus), streptococcal antigens, mycoplasma, mycobacterium, and herpes viruses. Highly preferred toxins are anthrax toxin (lethal factor, edema factor and protective antigen), SEA, SEB, SE1-3, SED and SEE.
[0079]The disease or condition may be considered to be an autoimmune process, for example rheumatoid arthritis, multiple sclerosis, lupus erythematosus, diabetes mellitus, myasthenia gravis, autoimmune thyroiditis, scleroderma, dermatomyositis, pemphigus, and other similar processes. Examples of such model systems for autoimmune diseases which can be used to evaluate the effects of the compounds and methods of the present invention are systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, insulin dependent diabetes mellitus, and experimental allergic encephalomyelitis. The procedures for conducting these experiments are presented in Clark et al., (1994) U.S. Pat. No. 5,284,935, the contents of which are incorporated herein by reference.
[0080]The disease or condition may be considered to be an allergic process, for example asthma, hayfever, allergic rhinitis, topical dermatitis, colitis, and other such processes initiated or associated with particular allergens or no defined allergen. Examples of such allergens are plant, animal, bacterial, parasitic allergens and metal-based allergens that cause contact sensitivity. Preferred allergens for use in the present invention are weed, grass, peanut, mite, flea and cat antigens.
[0081]Alternatively, the disease or condition may be a proliferative or malignant process, for example cancer, benign prostatic hypertrophy, psoriasis, adenomas or other cellular proliferations of intrinsic origin, or in response to a viral or other infectious, irritative, or environmental process.
[0082]The term `mammal` as used herein is meant to encompass the human species as well as all other mammalian species. The compounds and methods of this invention may be applied in the treatment of diseases and conditions occurring in individuals of all mammalian species. The term `individual` as used herein refers to one of any mammalian species, including the human species. The diseases and conditions occurring in individuals of the human species, and mentioned herein by way of example, shall include comparable diseases or conditions occurring in another species, whether caused by the same organism or pathogenic process, or by a related organism or pathogenic process, or by unknown or other known, organism and/or pathogenic process. The term `physician` as used herein also encompasses veterinarians, or any individual participating in the diagnosis and/or treatment of an individual of a mammalian species.
[0083]The present invention also provides for the administration of a compound, as a drug, a prodrug of the compound, or a drug-metabolite of the compound, in a suitable pharmaceutical formulation. The terms `administration of` or `administering a` compound is understood to mean providing a compound of the invention, as a drug, a prodrug of the compound, or a drug-metabolite of the compound, to an individual in need of treatment or prevention of a disease. Such a drug which contains one or more of the hybrid polypeptides of the present invention, as the principal or member active ingredient, for use in the treatment or prevention of one or more of the above-noted diseases and conditions, can be administered in a wide variety of therapeutic dosage forms in the conventional vehicles for topical, oral, systemic, and parenteral administration. The route and regimen of administration will vary depending upon the disease or condition to be treated, and is to be determined by the skilled practitioner. For example, the compounds can be administered in such oral dosage forms for example as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (either by bolus or infusion methods), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form. All of these is forms are well known to those of ordinary skill in the pharmaceutical arts.
[0084]The daily dose of the products may be varied over a range from 0.001 to 1,000 mg per adult per day. For oral administration, the compositions are preferably provided in the form of tables containing from 0.001 to 1,000 mg, preferably 0.001, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 10.0, 20.0, 50.0, 100.0 milligrams of active ingredient for the symptomatic adjustment of dosage according to signs and symptoms of the patient in the course of treatment. An effective amount of drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 50 mg/kg of body weight per day. The range is more particular from about 0.0001 mg/kg to 7 mg/kg of body weight per day.
[0085]Advantageously, suitable formulations of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses for example of two, three, or four times daily. The enhancing hybrid polypeptide of the present invention may be used to prepare a medicament or agent useful for the treatment of the diseases or conditions listed above. Furthermore, compounds of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regimen.
[0086]For treatment and prevention of disease, the hybrid polypeptide of the present invention may be administered in a pharmaceutical composition comprising the active compound in combination with a pharmaceutically acceptable carried adopted for topical administration. Topical pharmaceutical compositions may be, for example, in the form of a solution, cream, ointment, gel, lotion, shampoo, or aerosol formulation adapted for application to the skin. These topical pharmaceutical composition containing the compounds of the present invention ordinarily include about 0.005% to 5% by weight of the active compound in admixture with a pharmaceutically acceptable vehicle.
[0087]For the treatment and prevention of disease and conditions, for example listed above, the hybrid polypeptide of the present invention may be used together with other agents known to be useful in treating such diseases and conditions. For combination treatment with more than one active agent, where the active agents can be administered concurrently, the active agents can be administered concurrently, or they can be administered separately at staggered times.
[0088]The dosage regimen utilizing the compositions of the present invention is selected in accordance with a variety of factors, including for example type, species, age, weight, sex and medical condition of the patient, the severity of the condition to be treated, and the particular compound thereof employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the disease or condition. Optimal precision in achieving concentration of drug with the range that yields efficacy either without toxicity or with acceptable toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This process involves a consideration of the distribution, equilibrium, and elimination of the drug, an is within the ability of the skilled practitioner.
[0089]In the methods of the present invention, the compounds herein described in detail can form the active ingredient and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carders (collectively referred to herein as `carder materials`) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups, and the like, and consistent with conventional pharmaceutical practices. For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, aga, bentonite, xanthan gum and the like.
[0090]The liquid forms may be suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl cellulose and the like. Other dispersing agents which may be employed are glycerin and the like. For parental administration, sterile suspensions an solutions are desired. Isotonic predations which generally contain suitable preservatives are employed when intravenous administration is desired.
[0091]Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, for example, alcohols, aloe vera gel, allatoin, glycerine, vitamins A or E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, for example, alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
[0092]The hybrid polypeptide of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilameller vesicles and multilamellar vesicles. Liposomes can be formed from a variety of compounds, including for example cholesterol, stearylamine, and various phosphatidylcholines.
[0093]The hybrid polypeptide or formulation thereof of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihyrdo-pyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.
[0094]The hybrid polypeptides of the present invention and formulations thereof can be prepared using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are themselves known to those of ordinary skill in this art, but are not mentioned in greater detail herein.
[0095]As an alternative to administering the enhancing hybrid of the present invention directly to an individual to enhance the MHC Class II presentation of an antigenic epitope to T lymphocytes of the individual, a population of antigen presenting cells may be obtained from the individual and treated ex vivo with the enhancing hybrid of the present invention. These cells are treated with the enhancing hybrid under conditions appropriate for binding of the hybrid to an MHC Class II molecule of the antigen presenting cells. Once treated, the antigen presenting cells are administered to the individual under conditions which promote physical contact of the treated cells with T lymphocytes of the individual. As described above, the effect on the immune response, enhancement or suppression, will depend upon which subset of T cells are preferentially stimulated by the enhancing hybrid. Enhancement of the immune response may have a favorable effect upon the cytotoxic response against, for example, either a cancer cell or an infectious organism. Alternately, enhancement of the T suppressor cell response may have the effect of suppressing the immune response to a specific molecule. Such suppression may have a therapeutic effect when utilizing antigenic epitopes from etiological antigens of autoimmune diseases, for example, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, or lupus erythematosus. The methods and procedures for the ex vivo treatment of cells from a patient with the compounds and methods of the present invention may be adapted from the following patents, the contents of which are incorporated herein by reference: Rosenberg (1998) U.S. Pat. No. 5,126,132; Chada et al., (1997) U.S. Pat. No. 5,693,522; Kriegler et al., (1998) U.S. Pat. No. 5,849,586; Gruber et al., (1999) U.S. Pat. No. 5,856,185; and Kriegler et al., (1999) U.S. Pat. No. 5,874,077.
[0096]In another respect, the compounds and methods of the present invention can be used under ex vivo conditions to promote the generation of cytotoxic T lymphocytes, using the compounds and methods described in Celis et al., (1998) U.S. Pat. No. 5,846,827, the contents of which are incorporated herein by reference.
[0097]As discussed above, a non-comprehensive discussion of specific examples of epitopes/determinants useful as elements in the enhancing hybrids of the present invention is provided in the Exemplification section. Also found in the corresponding Exemplification section is a discussion of methods for using an enhancing hybrid containing such an element. One skilled in the art, through the application of no more than routine experimentation, can incorporate experimentally-determined or predicted epitopes/determinants into an enhancing hybrid for application to a wide range of disease or conditions.
[0098]In another aspect this invention relates to a method to identify and exploit naturally occurring Ii-Key/MHC Class II antigenic epitopes which have in the sequence a primary sequence motif which functions during the processing and binding of such peptides to MHC Class II molecules in the classical exogenous pathway, as does the synthetic Ii-Key/antigenic epitope hybrids.
[0099]Given the identification of the presence or absence of such Ii-Key motifs comprising, one can modify the amino acid sequence of the protein in a manner to introduce such a motif when one was not present, or to delete such a motif when one was present. Such modifications are obtained for example trough manipulation of the genes coding of the antigenic protein in a manner to substitute a functionally accepted amino acid in the Ii-Key motif. In some instances a deletion or insertion of amino acids can obtain the same end, for example when the antigenic epitope occurs at or near the N-terminus of the protein. Such modifications to change the immunogenecity of the protein have favorable clinical properties. For example, vaccine promoters can behave increased potency. Certain therapeutic proteins can have decrease immunogenecity.
[0100]In another aspect, the present invention relates to methods for selecting biologically active MHC Class II-presented epitopes and altering the immune response to such epitopes in antigenic proteins or polypeptides. Specifically, this disclosure provides method to identify in the amino acid sequence of a protein the presence or absence of a Ii-Key immunoregulatory motif of 5 amino acids preceding an experimentally determined or algorithm-predicted, MHC Class II-presented, antigenic epitope. This immunoregulatory Ii-Key motif enhances charging of the antigenic epitope, which follows it into the antigenic peptide binding site of MHC Class II molecules. Given predictions of antigenic epitopes within a protein, identifying the subset of those epitopes preceded by an Ii-Key motif improves greatly the efficiency of vaccine peptide selection. Also, by modifying the sequence of a protein or polypeptide, for example, either to introduce or to eliminate an Ii-Key motif before selected MHC Class II-presented epitopes, the immunological response to that protein can be altered.
[0101]Adverse immunological responses to a therapeutic protein can limit the use of such a protein. Such adverse immunological responses can be lessened either by decreasing immunogenecity of some of the MHC Class II-presented epitopes or by inducing immunosuppression. For either case, insertion or alteration(s) at the location of an Ii-Key motif appropriately spaced before an MHC Class II epitope can achieve that endpoint without alteration of the MHC Class II epitope itself. It may not be possible to alter residues within the MHC Class II-epitope without loss of the biological function of the therapeutic protein. The following procedure is followed in designing sequence modifications in a therapeutic protein of interest to alter its immunogenecity and/or the immune response to that protein.
[0102]The sequence of a protein, or a fragment thereof, is established by one of several methods. The protein or fragment thereof can be experimentally sequenced, or the sequence can be deduced from either the sequence of either the gene coding for the protein or a cDNA created from the RNA coding for the protein. Given that primary amino acid sequence, the experimentally determined or algorithm-predicted MHC Class II epitopes are specified. The experimentally determined epitopes are known from prior investigations. The algorithm-predicted epitopes are found by several methods, such as the ProPred MHC Class-II Binding Peptide Prediction Server (Raghava G P. Nat Biotechnol. 1999 17:555-61); Singh, H. Bioinformatics 2001 17:1236-7 (access via: http://www.imtech.res.in/raghava/propred/index.html)). An alternative program is the SYFPEITHI program (Rammensee H-G. Immunogenetics 1999 50: 213-219 (access via: http://134.2.96.221/scripts/MHCServer.dll/Ep.html)). These epitopes are also characterized with respect to the MHC Class II alleles, which are either known or predicted to present them to the immune system of humans or an experimental animal such as the mouse. Thus, differing sets of predicted epitopes are obtained, according to the relevant presenting MHC Class II allele. Some epitopes are presented by multiple MHC Class II alleles and are, therefore, preferred.
[0103]This disclosure presents a method for the identification of an Ii-Key immunoregulatory motif. Specifically, in the sequence of a protein, the immunoregulatory, Ii-Key motif is a segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, where that contiguous 5 amino acid segment is separated by 5 to 11 amino acids from the N-terminal residue of the MHC Class II-presented epitope.
[0104]The subset of such antigenic epitopes with the presence of an appropriately spaced Ii-Key motif lead to vaccine peptides to enhance the potency of the CD4+ T cell immune response. Such epitopes are considered to be more likely to be dominant or biologically active. Peptides with such epitopes are favored as vaccine protect against infectious diseases and cancer, and to immunosuppressive vaccines to allergy. The compositions and methods of the present invention relate to non-naturally occurring proteins or polypeptides which contain: 1) a C-terminal element comprising an MHC Class II-presented epitope; 2) an N-terminal element comprising an Ii-key motif; and 3) an intervening element comprising a sequence from about 4 to about 11 amino acid residues. The use of the term non-naturally occurring is intended to require that the protein or polypeptide is modified. Generally, the modification is by recombinant DNA techniques, and the modification or modifications take place within elements 2) or 3) as defined above. The designations "N- and C-terminal" are meant to refer only to the relationship of these elements in the 3-part segment specifically recited. One of skill in the art will recognize that if such a 3-part segment is located within a protein, it is likely that additional residues will extend in the C-terminal direction from the C-terminal element, and in the N-terminal direction from the N-terminal element. In addition to proteins or polypeptides as described above, the present application is also directed toward expressible nucleic acid sequences which encode such proteins or polypeptides.
[0105]In preferred embodiments, the non-naturally occurring protein or polypeptide is a modified form of a naturally occurring protein or polypeptide. Therapeutic proteins represent a particularly important class. Such modified proteins or polypeptides stimulate an immune response which differs from that induced by their non-modified, naturally-occurring counterparts. Such products include therapeutic proteins, such as hormones, cytokines, or other molecules interacting with cell surface receptors. Modifications of an Ii-Key motif can be made to eliminate its function, or a site N-terminal to a putative antigenic epitope can be modified to introduce an Ii-Key motif. Such modifications suppress a deleterious immune response to the therapeutic protein. Such products include the therapeutic protein, and fragments thereof, and genetic constructs leading to their expression.
[0106]Modifications most likely not to disturb the biological function of a therapeutic protein to be engineered to alter immunogenecity include the following. Presence is scored of sequences, and even individual amino acid residues, which are known from the crystallographic structure of the protein to be superficially exposed on the protein, and thus more likely to accept a mutation without loss of function. In the case of therapeutic proteins for which the three dimensional structure has not been determined, various methods are applied to predicting acceptance of mutations to engineer an Ii-Key box appropriately spaced from an antigenic epitope. Distances from the N-terminus and from the C-terminus of the protein are determined. Upon modestly denaturing conditions, N-terminal and C-terminal antigenic epitopes can be presented by MHC Class II molecules. Epitopes at the N-terminus of the protein are favored over epitopes at the C-terminus, in part because the to-be-designed Ii-Key box is more distal. Also sequence alterations are more likely to be accepted in a sequence, which is predicted to be on the surface of the protein, preferably in relatively loose configuration. Such segments can be identified in homologous proteins with a relatively higher frequency of naturally occurring mutations. Segments are identified containing residues, which in site-directed mutational studies, have been shown to accept amino acid substitutions. By the preceding and additional methods, one skilled in the art will predict segments of a protein that are more likely to accept without loss of function, amino acid substitutions at residue positions resulting in the creation of Ii-Key box motifs at appropriate N-terminal displacements from the N-terminus of an antigenic epitope. The following peptide sequences are targeted for Ii-Key box manipulations, in rank order: epitopes known to be MHC Class II-presented, epitopes predicted to be MHC Class II-presented by MHC Class II alleles present either in the highest frequency among humans or in the animal strain of experimental interest. Some of these methods are presented in U.S. Pat. No. 5,679,527 (1997), the content of which is incorporated herein by reference.
[0107]In addition to the above site-specific engineered replacements, one skilled in the art will use additional combinatorial molecular biological methods to generate mutations within sets of residue positions to create an Ii-Key box motif spaced 4 to 8 amino acids N-terminal to a selected, either known or putative antigenic epitope. Such methods may encompass the preparations of multiple products, which are screened for altered immunogenecity with or without retention of biological activity.
[0108]Each experimentally determined or algorithm-predicted epitope in the protein of interest is examined for the presence in its primary sequence of a segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, where that contiguous 5 amino acid segment is separated by 4 to 12 amino acids from the N-terminal residue of a MHC Class II-presented epitope. The subset of all experimentally or algorithm-predicted epitopes meeting these criteria are preferred for developmental work, including for example synthesis of peptide vaccines, chemical modifications of those vaccines for a favorable therapeutic effect, experimental study in animals, and clinical studies. In this disclosure the standard single letter nomenclature of the International Union of Pure and Applied Chemists is used to identify amino acids within the sequence of proteins or peptides.
[0109]More preferable is the set of experimentally or algorithm-predicted epitopes with a 4 to 8 amino acids separation from the N-terminal residue of the MHC Class II-presented epitope, of the segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg. This set constitutes a subset of the set of experimentally or algorithm-predicted epitopes with a 0 to 12 amino acids separation from the N-terminal residue of the MHC Class II-presented epitope, of the segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg. An important utility in this method is the reduction in the number of candidate epitopes subject to study toward therapeutic or diagnostic development objectives. In each of the Exemplification Tables of this disclosure presenting either experimentally determined or algorithm-predicted epitopes, the presence of an Ii-Key box separated by 4-10 amino acids from the N-terminus of the MHC Class II-presented epitope is indicated, with the number of intervening residue positions. Among these, preference is ranked according to the length of the separating interval, with shorter being better, among spacers of 4 or more amino acids.
[0110]Peptides which are chosen for synthesis have within the natural primary sequence of the protein, an MHC Class II-presented epitope and a segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, the intervening segment of 0 to 12 amino acids, and the antigenic epitope, comprising a total length of 12 to 34 amino acids. More preferred is a synthetic peptide synthesized according to the primary sequence of the protein, including the segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, the intervening segment of 4 to 11 amino acids, and the antigenic epitope, comprising a total length of 15 to 25 amino acids. The sequence of the synthetic peptide will usually be the natural sequence of a mammalian protein, but also the protein may be a non-natural sequence, such as that generated by methods using a combinatorial library with selections for a useful function. Furthermore, the sequence of the protein may include modifications of the sequence of natural protein, including for example substitution, insertion, or deletion of one or more amino acids, including the use of non-natural amino acids.
[0111]The selected peptides, including the segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, the intervening segment of 4 to 8 amino acids, and the antigenic epitope, comprising a total length of 12 to 34 amino acids will be modified to obtain favorable biological and pharmacokinetic properties. These medications are selected from the group consisting of: a) acetylation of the N-terminus, b) amidation of the C-terminus; c) replacement of an amino acid with another natural or synthetic amino acid, d) replacement of an L-amino acid with a D-amino acid, e) inversion of the amino acid sequence and use of D-amino acids in each residue positions, f) modifications to limit proteolysis or clearance (inactivation), and g) modifications to improve solubility, transport and half-life. Methods of chemical modification of therapeutic peptides for favorable therapeutic properties are presented, for example, in U.S. Pat. No. 5,679,527, the disclosure of which is incorporated herein by reference.
[0112]The method to design such modifications start with a list of identified epitopes, ranked according to each of several characteristics in order to identify segments of the therapeutic protein, which are more likely to accept without loss of function, amino acid substitutions which create an Ii-Key box motif appropriately spaced from the N-terminus of an antigenic epitope. The characteristics by which the epitopes are ranked include, without limitation, the following. Presence is scored of sequences, and even individual amino acid residues, which are known from the crystallographic structure of the protein to be superficially exposed on the protein, and thus more likely to accept a mutation without loss of function. In the case of therapeutic proteins for which the three dimensional structure has not been determined, various methods are applied to predicting acceptance of mutations to engineer an Ii-Key box appropriately spaced from an antigenic epitope. Distances from the N-terminus and from the C-terminus of the protein are determined. Upon modestly denaturing conditions, N-terminal and C-terminal antigenic epitopes can be presented by MHC Class II molecules. Epitopes at the N-terminus of the protein are favored over epitopes at the C-terminus, in part because the to-be-designed Ii-Key box is more distal. Presence in a sequence motif, which is predicted to be on the surface of the protein, preferably in relatively loose configuration. Segments are identified which in homologous proteins have a relatively higher frequency of naturally occurring mutations. Segments are identified containing residues, which in site-directed mutational studies have been shown to accept amino acid substitutions. By the preceding and additional methods, one skilled in the art will predict segments of a protein which are more likely to accept without loss of function, amino acid substitutions at residue positions which create Ii-Key box motifs at appropriate N-terminal displacements from the N-terminus of an antigenic epitope which is highly ranked according to the following ranking scheme: epitopes known to be MHC Class II-presented, epitopes predicted to be MHC Class II-presented by MHC Class II alleles present either in the highest frequency among humans or in the animal strain of experimental interest. Some of these methods are presented in U.S. Pat. No. 5,679,527 (1997) the disclosures of which are incorporated herein by reference.
[0113]The Ii-Key box/spacer identifying algorithm is applied within the amino acid sequence of the protein to examine regions N-terminal to each of the above experimentally determined or predicted MHC Class II-presented epitopes, in a manner to identify three categories: a) presence of an Ii-Key box motif spaced by 4 to 8 amino acids, N-terminal to the antigenic epitope, b) presence of an Ii-Key box motif spaced by 4 to 8 amino acids, N-terminal to the antigenic epitope if one or more amino acids were exchanged for a member of the group Leu, Ile, Val, Phe, Met and/or one or more amino acids were exchanged for a member of the group His, Lys and Arg in the primary sequence.
[0114]In addition to the above site-specific engineered replacements, one skilled in the art will use additional combinatorial molecular biological methods to generate mutations within sets of residue positions to create an Ii-Key box motif spaced 4 to 8 amino acids N-terminal to a selected, either known or putative antigenic epitope. Such methods may encompass the preparations of multiple products, which are screened for altered immunogenecity with or without retention of biological activity.
[0115]Many uses of Ii-Key antigenic epitope hybrids can be described with respect to individual antigenic proteins. Such uses are presented in the Examples, in varying degrees of detail. The concepts, which are presented in the context of one Example, apply nevertheless in the cases of all Examples when appropriate, even when they are not repeated in the context of each individual Example. While such specific examples well present methods to design and synthesize Ii-Key antigenic epitope hybrids of specific proteins by which such Ii-Key antigenic epitope hybrids can be created and used with respect to other proteins of interest, as the need might arise from to time.
[0116]In another aspect, this invention relates to the use of Ii-Key/antigenic epitope hybrids to enhance protective immune responses to a subsequently administered DNA vaccine or against an attenuated infectious pathogen vaccine. Such adjuvant vaccine preparations can be referred to as PreVaccines®. One example is the use of Ii-Key antigenic epitope hybrids in vaccination protocols to protect against variola. Uses in protecting against smallpox virus are considered in relatively greater detail in a corresponding section of the Exemplification section which follows. Considerations detailed herein also serve to model applications directed toward other pathogens. In the case of smallpox vaccination, Ii-Key antigenic epitope hybrids are used to elicit a Th1 response to one or more MHC Class II-presented epitopes of the gp42 extracellular envelope protein coded by the B5R viral gene of vaccinia. Individuals so vaccinated will have an anamnestic response which is more rapid and of higher potency in terms of antibody titers and isotype an affinity maturation, CTL and memory responses to challenge by cDNA vaccines for the B5R gene, by vaccinia, or by variola. In a related application, such PreVaccines® can be used before vaccination with recombinant vaccinia virus containing either Ii-RGC genes or CIITA plus Ii-RGC genes. The recombinant vaccinia virus containing an Ii-RGC gene, upon infection within a professional antigen presenting cell such as a dendritic cell, will lead to MHC Class II-restricted T helper cell responses in those cells as described. In the case of recombinant vaccinia virus containing both an Ii-RGC gene and a CIITA gene, such a virus upon infecting cells which do not normally express MHC Class II molecules, such as dendritic cells, will express MHC Class II molecules without Ii protein. A wide repertoire of MHC Class II-presented epitopes are thus represented and the response to those epitopes is further enhanced by prior expansion of responses to the MHC Class II epitope in the PreVaccine®. Such a use can be further augmented by prior immunization of mammals with Ii-Key antigenic epitope hybrids in an appropriate dose, vehicle, route and schedule. Ii-Key/antigenic epitope hybrids can thus be used either as a stand-alone protective vaccine or as a PreVaccine® used in conjunction with vaccines for other viruses and infectious pathogens, for example, without limitation, HIV, Bacillus anthracis, EBOLA virus and Marburg virus.
EXEMPLIFICATION
Example 1
Ii-Key/Ara h 1 Antigenic Epitope Hybrids
[0117]In one aspect this invention relates to therapeutic modulation of pathological allergic responses of some humans to peanuts and other edible nuts. Such responses include potentially fatal asthmatic or anaphylactic reactions. Good progress has been made in identifying and sequencing the principal protein allergens in peanuts and other nuts mediating these pathological responses. Crossed-radioimmunoelectrophoresis has identified 16 allergenic fractions in raw peanut and sodium dodecylsulfate polyacrylamide gel electrophoresis has revealed 32 protein bands (Barnett D. J Allergy Clin Immunol. 1983 72:61-68). Three major allergens have been identified. Ara h 1 of 64.5 kDa is a member of the vicilin family of seed storage proteins (Burks A W. J Allergy and Clin Immunol. 1991 88:172-9). Ara h 2 of 17.5 kDa is a member of the conglutin family of seed storage proteins (Burks A W. J Allergy and Clin Immunol. 1992 90:962-9). Ara h 3 of 60 kDa, a preproglobulin, is a member of the glycinin-like seed storage proteins (Rabjohn P. J Clin Invest. 1999 103:535-42). For Ara h 1, 23 IgE-recognized epitopes have been mapped, with 4 being dominant. For Ara h 2, 10 IgE-recognized epitopes have been mapped, with 3 being dominant. For Ara h 3, 4 IgE-recognized epitopes have been mapped, with 1 being dominant. For each of these three allergens, the respective cDNAs have been isolated and expressed. The deduced protein sequences are presented below (Tables 1.1, 2.1 and 3.1).
[0118]Development of allergy-inducing IgE antibodies is regulated by a subset of CD4+ T cells, the receptors of which recognize antigenic peptides presented by MHC Class II-molecules. The recognition of such epitopes by CD4+ T cells can lead either to a Th1 response, in which the responding T cells are characterized by synthesis of predominantly certain cytokines such as IFN-γ, or to a Th2 response, in which the responding T cells are characterized by synthesis of predominantly other cytokines such as IL-4 and IL-10. In patients with allergen-induced asthma, a Th2 pattern of response enhances synthesis of IgE molecules recognizing many different surface epitopes of the offending allergen(s). Binding of IgE to such allergens activates a cascade of biological mediators resulting in the asthmatic symptoms. The compounds and methods of the invention can be applied to the modification of responses in a Th1 or Th2 pathway-specific manner to obtain clinically desired effects. Such modifications can be illustrated for the control of asthma.
[0119]In animal studies of asthmatic allergic responses to protein antigens, it was discovered that substitution of one or more amino acids within the MHC Class II antigenic epitope leads to potential therapeutic agents inducing an altered T cell immune response. Specifically, such altered antigenic peptides modified a predominantly Th2 response, which promotes asthmatic responses, to a predominantly Th1 response (Janssen E. J Immunol. 2000 164:1580-8; Janssen E M. J Immunol. 2000 165:7207-14). Such immunodeviation from a Th2 to a Th1 pattern functionally suppresses the asthmatic response. However replacement of individual amino acids in a MHC Class II-presented epitope of an offending allergen is expected to alter potency of binding of the antigenic peptides in the antigenic peptide binding site as well as the repertoire of T cell receptors responding to the antigenic peptide. Affinity of the antigenic epitope peptide for a patient's MHC Class II alleles can be decreased by such structural manipulations. One significant advantage of the method of this invention is the ability to immunodeviate the pattern of Th subset activation from the Th2 pathway to the Th1 pathway, without changing the sequence of the antigenic epitope. Since MHC Class II molecules demonstrate allele-specific preferences for some antigenic peptides and not for other antigenic peptides (which might nevertheless be well presented by other MHC Class II alleles), there is no issue of potentially decreased potency of Ii-Key/antigenic epitope hybrids. In fact, given the increase in potency of presentation of epitopes within Ii-Key/antigenic epitope hybrids, one can expect presentation by a wider range of MHC Class II alleles. Another clinically preferred characteristic of the Ii-Key/antigenic epitope hybrids over sequence-modified antigenic epitope peptides is that the dose required to achieve immunodeviation is much less (by a factor of 10 to 100) and therefore potentially fatal anaphylaxis is much less likely to occur.
[0120]In another aspect, this invention relates to the design of Ii-Key/Ara h 1 antigenic epitope hybrids. Such Ii-Key/Ara h 1 antigenic epitope hybrids comprise the Ii-Key motif LRMK (SEQ ID NO: 3) and acceptable modifications, linked through a simple, flexible linker to a MHC Class II-presented epitopes of the Arachis hypogaea 1 (Ara h 1) major allergen protein found in peanuts and some additional edible nuts. The amino acid sequence of this allergen (626 amino acids) is presented in Table 1.1. The sequence of Ara h 1 was taken from GenBank entry gi/11683gi/ allergen Ara h 1. MHC Class II-presented epitopes within this protein sequence were identified with the Singh ProPred MHC Class-II Binding Peptide Prediction Server (Raghava G P. Nat Biotechnol. 1999 17:555-61; Singh, H. Bioinformatics 2001 17:1236-7 (access via: http://www.imtech.res.in/raghava/propred/index.html)). The ProPred program evaluates sequences for presentation by many common MHC Class II alleles. An alternative program is the SYFPEITHI program (Rammensee H-G. Immunogenetics 1999 50: 213-219 (access via: http://www.uni-tuebingen.de/uni/kxi/)). Epitopes with highest scores were identified for their presentation by 51 HLA-DR alleles, that cover more than 90% of the MHC Class II alleles. The highest scoring epitopes predicted with the ProPred program are likely to be experimentally antigenic. The peptides listed in Table 1.2 have the highest scoring epitopes, in the ProPred program analysis for Ara h 1. Ii-Key/Ara h 1 hybrids containing some of the predicted MHC Class II-presented Ara h 1 epitopes of Table 1.2 are listed in Table 1.3. Experimentally defined IgE-binding Ara h 1 epitopes which overlap with predicted MHC Class II-presented Ara h 1 epitopes are listed in Table 1.4. Ii-Key/Ara h 1 hybrids containing predicted MHC Class II Ara h 1 epitopes and experimentally determined IgE-binding Ara h 1 epitopes are listed in Table 1.5.
TABLE-US-00001 TABLE 1.1 Deduced amino acid sequence of Ara h 1. 1 mrgrvsplml llgilvlasv sathaksspy qkktenpcaq rclqscqqep (SEQ ID NO: 10) 51 ddlkqkaces rctkleydpr cvydprghtg ttnqrsppge rtrgrqpgdy 101 dddrrqprre eggrwgpagp rerereedwr qpredwrrps hqqprkirpe 151 gregeqewgt pgshvreets rnnpfyfpsr rfstrygnqn grirvlqrfd 201 qrsrqfqnlq nhrivqieak pntlvlpkha dadnilviqq gqatvtvang 251 nnrksfnlde ghalripsgf isyilnrhdn qnlrvakism pvntpgqfed 301 ffpassrdqs sylqgfsrnt leaafnaefn eirrvlleen aggeqeergq 351 rrwstrssen negvivkvsk ehveeltkha ksvskkgsee egditnpinl 401 regepdlsnn fgklfevkpd kknpqlqdld mmltcveike galmlphfns 451 kamvivvvnk gtgnlelvav rkeqqqrgrr eeeededeee egsnrevrry 501 tarlkegdvf impaahpvai nasselhllg fginaennhr iflagdkdnv 551 idqiekqakd lafpgsgeqv ekliknqkes hfvsarpqsq sqspsspeke 601 spekedqeee nqggkgplls ilkafn
TABLE-US-00002 TABLE 1.2 Predicted MHC Class II-presented epitopes of Ara h 1. PEPTIDE SEQ ID NO: Pos. Sequence Score Ii-Key NO: 1.2.1 417 V K P D K K N P Q 6.00 -- 11 1.2.2 193 I R V L Q R F D Q 6.00 -- 12 1.2.3 313 L Q G F S R N T L 6.00 -- 13 1.2.4 453 M V I V V V N K G 6.00 3 14 1.2.5 457 V V N K G T G N L 5.20 -- 15 1.2.6 498 V R R Y T A R L K 5.30 -- 16 1.2.7 209 L Q N H R I V Q I 5.30 8 17 1.2.8 206 F Q N L Q N H R I 4.40 5 18 1.2.9 9 M L L L G I L V L 5.30 3 19 1.2.10 11 L L G I L V L A S 5.50 4 20 1.2.11 1 M R G R V S P L M 4.25 -- 21 1.2.12 15 L V L A S V S A T 4.20 -- 22 1.2.13 429 L D M M L T C V E 5.10 9 23 1.2.14 264 L R I P S G F I S 5.00 5 24 1.2.15 270 F I S Y I L N R H 4.48 -/11 25 1.2.16 275 L N R H D N Q N L 4.10 6 26 1.2.17 325 F N A E F N E I R 4.30 -- 27 1.2.18 329 F N E I R R V L L 4.60 -- 28 1.2.19 335 V L L E E N A G G 4.20 -- 29
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
[0121]In Table 1.2, PEPTIDES: 1.2.1, 1.2.3, 1.2.6, 1.2.5, and 1.2.18 overlap to some degree with experimentally defined IgE-binding epitopes of Table 1.4. PEPTIDES 1.2.9, 1.2.10, 1.2.11, 1.2.12 are peptides with altered amino acid sequences in a recombinant, mutated Ara h 1 (Burks A W. Eur J. Immunol. 1997 245:334-9). IgE epitopes were defined further in the work of Shin et al. (J Biol. Chem. 1998 273:13753-9).
TABLE-US-00003 TABLE 1.3 Ii-Key/Ara h 1 hybrids containing some of the predicted MHC Class II-presented Ara h 1 epitopes of Table 1.2. SEQ ID PEPTIDE Pos. Sequence NO: 1.3.1 192 Ac-LRMK-ava-IRVLQRFDQ-NH2 30 1.3.2 1 Ac-LRMK-ava-MRGRVSPLM-NH2 31 1.3.3 1/8/10/14 Ac-LRMK-ava-MRGRVSPLML 32 LLGILVLASV SAT-NH2 1.3.4 205 Ac-LRMK-ava-FQNLQNHRI-NH2 33 1.3.5 205/208 Ac-LRMK-ava-FQNLQNHRIVQI- 34 NH2 1.3.6 428 Ac-LRMK-ava-LDMMLTCVE-NH2 35 1.3.7 263 Ac-LRMK-ava-LRIPSGFIS-NH2 36 1.3.8 263/269/274 Ac-LRMK-ava- 37 LRIPSGFISYILNRHDNQNL-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
[0122]The activity of additional Ii-Key/Ara h antigenic epitope hybrids are tested with one residue of a-aminovaleric acid as a spacer because, in previous studies of a series of hybrids with systematic variation of spacer structures, the hybrid with one ava residue was no less active than any hybrid with a more complex spacer sequence. In the Ara h hybrids, the Ii-Key-spacer (LRMK-ava) (SEQ ID NO: 9) sequence was linked to the first amino acid of the ProPred-identified peptide, which amino acid is thought to fit into pocket 1 of the antigenic peptide-binding site of the MHC Class II molecules.
[0123]The peptides of Table 1.3 are characterized as follows. PEPTIDE 1.3.1 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 1.2.2. PEPTIDE 1.3.2 is a composite of the first two MHC Class II-presented epitopes (PEPTIDE 1.2.9; PEPTIDE 1.2.11), overlapping by two amino acids. PEPTIDE 1.3.3 is a composite of the first four MHC Class II-presented epitopes (PEPTIDE 1.2.11, PEPTIDE 1.2.9, PEPTIDE 1.2.10, PEPTIDE 1.2.12). PEPTIDES 1.3.2 and 1.3.3 are peptides with altered amino acid sequences in the recombinant, mutated Ara h 1 (Burks A W. Eur J Immunol. 1997 245:334-9). PEPTIDE 1.3.4 contains the ProPred-predicted MHC Class II-presented epitopes PEPTIDE 1.2.8. PEPTIDE 1.3.5 is the composite of two MHC Class II-predicted epitopes (PEPTIDE 1.2.7 and PEPTIDE 1.2.8), overlapping by six amino acids. PEPTIDE 1.3.6 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 1.2.13. PEPTIDE 1.3.7 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 1.2.14. PEPTIDE 1.3.8 is the composite of three MHC Class II-predicted epitopes (PEPTIDE 1.2.14, PEPTIDE 1.2.15 and PEPTIDE 1.2.16), overlapping by three and four amino acids, respectively.
TABLE-US-00004 TABLE 1.4 Experimentally defined IgE-binding Ara h 1 epitopes which overlap with predicted MHC Class II-presented Ara h 1 epitopes. PEPTIDE Pos. Sequence SEQ ID NO: 1.4.1 409 NNFGKLFEVK 38 1.4.2 311 SYLQEFSRNT 39 1.4.3 498 RRYTARLKEG 40 1.4.4 325 FNAEFNEIRR 41 1.4.5 461 GTGNLELVAV 42
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00005 TABLE 1.5 Ii-Key/Ara h 1 hybrids containing predicted MHC Class II Ara h 1 epitopes and experimentally determined IgE-binding Ara h 1 epitopes. SEQ ID PEPTIDE Pos. Sequence NO: 1.5.1 416 Ac-LRMK-ava-NNFGKLFEVKPDKKNPQ- 43 NH2 1.5.2 312 Ac-LRMK-ava-LQGFSRNTL-NH2 44 1.5.3 496 Ac-LRMK-ava-VRRYTARLK-NH2 45 1.5.4 452 Ac-LRMK-ava-MVIVVVNKG-NH2 46 1.5.5 456 Ac-LRMK-ava-VVNKGTGNL-NH2 47 1.5.6 452 Ac-LRMK-ava- 48 MVIVVVNKGTGNLELVAV-NH2 1.5.7 324 Ac-LRMK-ava-FNAEFNEIR-NH2 49 1.5.8 328 Ac-LRMK-ava-FNEIRRVLL-NH2 50 1.5.9 334 Ac-LRMK-ava-VLLEENAGG-NH2 51 1.5.10 324/ Ac-LRMK-ava- 52 328/ FNAEFNEIRRVLLEENAGG-NH2 334
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the proposed hybrid containing a predicted MHC Class II-presented epitope of Table 1.2 and an IgE binding epitope of Table 1.4. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
[0124]The PEPTIDES of Table 1.5 are characterized as follows. PEPTIDES 1.5.1, 1.5.6, and 1.5.10 include residues of an experimentally defined, IgE-binding epitope. PEPTIDES 1.5.1, 1.5.2, 1.5.4, 1.5.6, 1.5.9, and 1.5.10 have residues of a ProPred-predicted MHC Class II-presented epitopes. PEPTIDES 1.5.2, 1.5.3, 1.5.4, 1.5.5, 1.5.6, 1.5.7, 1.5.8 and 1.5.10 share amino acids between overlapping IgE binding and MHC Class II-presented epitopes. PEPTIDES 1.5.4, 1.5.5, 1.5.6, 1.5.8, 1.5.9, and 1.5.10 share amino acids between overlapping MHC Class II-presented epitopes.
[0125]The peptides of Table 1.5 are characterized as follows. PEPTIDE 1.5.1 is the composite of MHC Class II-presented epitope with the highest ProPred predictive binding score (PEPTIDE 1.2.1) and IgE binding epitope (PEPTIDE 1.4.1), overlapping by 2 amino acids. PEPTIDE 1.5.2 is the composite of MHC Class II-presented epitope SEQ ID NO 44 and IgE binding epitope PEPTIDE 1.4.2, overlapping by 8 amino acids. PEPTIDE 1.5.3 is the composite of MHC Class II-presented epitope PEPTIDE 1.2.6 and IgE binding epitope PEPTIDE 1.4.3, overlapping by 8 amino acids. PEPTIDE 1.5.4 contains MHC Class II-predicted epitope PEPTIDE 1.2.4 and an IgE binding epitope PEPTIDE 1.4.5, overlapping by 1 amino acid. PEPTIDE 1.5.5 contains MHC Class II-predicted epitope PEPTIDE 1.5 and an IgE binding epitope PEPTIDE 1.4.5, overlapping by 5 amino acids. PEPTIDE 1.5.6 is the composite of the two MHC Class II-predicted epitopes, PEPTIDE 1.2.4 and PEPTIDE 1.2.5, overlapping by 5 amino acids. Additionally, there is a 5 amino acids overlap with IgE binding epitope (PEPTIDE 1.4.5). PEPTIDE 1.5.7 contains MHC Class II-predicted epitope PEPTIDE 1.2.17 and an IgE binding epitope PEPTIDE 1.4.4, overlapping by 9 amino acids. PEPTIDE 1.5.8 contains MHC Class II-predicted epitope PEPTIDE 1.18 and an IgE binding epitope PEPTIDE 1.4.4, overlapping by 6 amino acids. PEPTIDE 1.5.9 contains MHC Class II-predicted epitope PEPTIDE 1.2.19. PEPTIDE 1.5.10 is the composite of the three MHC Class II-predicted epitopes PEPTIDES 1.2.17, 1.2.18, and 1.2.19 and IgE binding epitope PEPTIDE 3.1.5. PEPTIDE 1.5.5 is the composite of three MHC Class II-predicted epitopes (PEPTIDES 1.2.17, 1.2.18 and 1.2.19), overlapping by 5 and 3 amino acids, respectively. Additionally, there is a 9 amino acid overlap with IgE binding epitope (PEPTIDE 1.4.4).
Example 2
Ii-Key/Ara h 2 Peanut Antigenic Epitope Hybrids
[0126]In another aspect, this invention relates to the design of Ii-Key/Ara h 2 antigenic epitope hybrids. Sampson, WO 0052154, a series of Ara h 2 MHC Class II-presented epitopes, which had been experimentally identified by Burks A W. (J Allergy Clin Immunol. 1992 90:962-7). Ara h 2-specific T cell lines were established from the peripheral blood of 12 atopic and 4 nonatopic individuals. All of the T cell lines were predominantly CD 4+ T cells. Reactivity of each of these T cell lines was tested against individual peptides from a library of overlapping Ara h 2 peptides. Four immunodominant T cell epitopes were identified for Ara h 2: epitope 1 (amino acids 18-28), epitope 2 (amino acids 45-55), epitope 3 (amino acids 95-108), and epitope 4 (amino acids 134-144). Epitopes 1, 2, and 4 have overlapping sequences with IgE antibody-recognized epitopes while epitope 3 does not overlap IgE binding epitopes. Bannon and colleagues suggested that such sequences provide for the possibility for the development of a non-anaphylactic, T cell-directed immunotherapeutic (Bannon G A. Int Arch Allergy Immunol. 2001 124:70-72). The sequence of Ara h 2 in Table 2.1 was taken from GenBank gi/15418705/allergen II [Arachis hypogaea]. Experimentally defined MHC Class II-presented Ara h 2 epitopes are listed in Table 2.2. Ii-Key/Ara h 2 hybrids containing some of the experimentally defined MHC Class II-presented Ara h 2 epitopes of Table 2.2 are listed in Table 2.3. Predicted MHC Class II epitopes of Ara h 2 are listed in Table 2.4. Ii-Key/Ara h 2 hybrids containing some of the predicted MHC Class II-presented Ara h 2 epitopes of Table 2.4 are listed in Table 2.5. Experimentally defined IgE-binding Ara h 2 epitopes, which overlap with predicted MHC Class II-presented Ara h 2 epitopes from Table 2.4 are listed in Table 2.6. Hybrids containing predicted MHC Class II Ara h 2 epitopes and overlapping experimentally determined IgE-binding Ara h 2 epitopes are listed in Table 2.7.
TABLE-US-00006 TABLE 2.1 Deduced amino acid sequence of Ara h 2. 1 makltilval alfllaahas arqqwelqgd rrcqsqlera nlrpceqhlm (SEQ ID NO: 53) 51 qkiqrdedsy erdpyspsqd pyspspydrr gagssqhqer ccnelnefen 101 nqrcmcealq qimenqsdrl qgrqqeqqfk relrnlpqqc glrapqrcdl 151 dvesgg
TABLE-US-00007 TABLE 2.2 Experimentally defined MHC Class II-presented Ara h 2 epitopes. SEQ ID PEPTIDE Pos. Sequence Ii-Key NO: 2.2.1 22 RQQWE LQGDRRCQSQ 3 54 2.2.2 42 LRPCEQHLMQKIQRDEDSYE -- 55 2.2.3 7 HQERCCNELN -- 56 2.2.4 102 QRCMCEALQQ -- 57 2.2.5 137 PQQCGLRAPQ -- 58
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
[0127]The PEPTIDES of Table 2.2 are characterized as follows. PEPTIDES 2.2.1, 2.2.2, 2.2.4, and 2.2.5 are ProPred-predicted MHC Class II-presented sequences. PEPTIDE 2.2.1 contains an IgE binding epitopes. PEPTIDES 2.2.1 and 2.2.2 have overlapping amino acids of the IgE binding epitope and MHC Class II-presented epitope. Pos. is the residue number in the primary amino acid sequence of the first amino acid of the epitope. Many of the experimentally predicted epitopes are also predicted with the ProPred algorithm, either entirely or partially.
TABLE-US-00008 TABLE 2.3 Ii-Key/Ara h 2 hybrids containing some of the experimentally defined MHC Class II-presented Ara h 2 epitopes of Table 2.2. SEQ ID PEPTIDE Pos. Sequence NO: 2.3.1 19 Ac-LRMK-ava-RQQWE LQGDRRCQSQ-NH2 59 2.3.2 39 Ac-LRMK-ava- 60 LRPCEQHLMQKIQRDEDSYE-NH2 2.3.3 84 Ac-LRMK-ava-HQERCCNELN-NH2 61 2.3.4 99 Ac-LRMK-ava-QRCMCEALQQ-NH2 62 2.3.5 135 Ac-LRMK-ava-PQQCGLRAPQ-NH2 63
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 2.2. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
[0128]The PEPTIDES of Table 2.3 are characterized as follows. PEPTIDE 2.3.1 contains an experimentally defined, IgE-binding epitope. PEPTIDES 2.3.1 and 2.3.2 share amino acids between overlapping IgE binding and MHC Class II-presented epitopes. PEPTIDES 2.3.2 and 2.3.3 are peptides with altered amino acid sequences in the modified Ara h 1 of Burks and colleagues (Burks A W. Eur J Immunol. 1997 245:334-9). Pos. is the residue number in the primary amino acid sequence of the first amino acid of the epitope.
TABLE-US-00009 TABLE 2.4 Predicted MHC Class II epitopes of Ara h 2. SEQ ID PEPTIDE Pos. Sequence Score Ii-key NO: 2.4.1 5 I L V A L A L F L 6.10 -- 64 2.4.2 26 L Q G D R R C Q S 5.80 8 65 2.4.3 3 L T I L V A L A L 5.30 -- 66 2.4.4 49 L M Q K I Q R D E 4.10 -- 67 2.4.5 12 L F L L A A H A S 3.30 4 68 2.4.6 7 L V A L A L F L L 4.70 -- 69 2.4.7 42 L R P C E Q H L M 3.60 -- 70 2.4.8 10 L A L F L L A A H 3.30 2 71 2.4.9 133 L R N L P Q Q C G 2.70 -- 72 2.4.10 37 L E R A N L R P C 2.20 -- 73 2.4.11 13 F L L A A H A S A 1.90 5 74 2.4.12 77 Y D R R G A G S S 1.90 -- 75 2.4.13 98 F E N N Q R C M C 1.70 -- 76
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
[0129]The PEPTIDES of Table 2.4 are characterized as follows. PEPTIDES 2.4.1, 2.4.5, 2.4.6, 2.4.8, and 2.4.11 are peptides not preserved in an Ara h 2 modified to decrease allergic IgE binding. In PEPTIDE 2.4.4 R54 is replaced by A. In PEPTIDE 2.4.7 P43 and Q46 are each replaced by A. PEPTIDES 2.4.2, 2.4.4, 2.4.9, 2.4.10, and 2.4.13 are experimentally defined T cell epitopes. PEPTIDES 2.4.2, 2.4.4, 2.4.7, 2.4.10, and 2.4.11 have amino acids of an IgE binding epitope.
TABLE-US-00010 TABLE 2.5 Ii-Key/Ara h 2 hybrids containing some of the predicted MHC Class II-presented Ara h 2 epitopes of Table 2.4. SEQ ID PEPTIDE Pos. Sequence NO: 2.5.1 5 Ac-LRMK-ava-ILVALALFL-NH2 77 2.5.2 3 Ac-LRMK-ava-LTILVALAL-NH2 78 2.5.3 6 Ac-LRMK-ava-LVALALFLL-NH2 79 2.5.4 3/5/6 Ac-LRMK-ava-LTILVALALFLL-NH2 80 2.5.5 132 Ac-LRMK-ava-LRNLPQQCG-NH2 81 2.5.6 76 Ac-LRMK-ava-YDRRGAGSS-NH2 82 2.5.7 97 Ac-LRMK-ava-FENNQRCMC-NH2 83
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 2.4. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
[0130]The PEPTIDES of Table 2.5 are characterized as follows. PEPTIDES 2.5.1, 2.5.2, 2.5.3, 2.5.4 are peptides not preserved in the modified Ara h 2. PEPTIDES 2.5.5 and 2.5.7 are experimentally defined CD4+ T cell epitopes.
[0131]In another aspect, this invention provides for the immunodeviation of an allergic patient's antibody response from an IgE pattern to an IgG or IgG subtype pattern. The decrease synthesis of IgE antibodies to the allergen and/or the synthesis of IgG antibodies, which block the binding of IgE antibodies, has a desired therapeutic effect. To this end MHC Class II epitopes of the allergen are joined with an IgE binding peptide sequence in an Ii-Key/MHC Class II epitope/IgE epitope hybrid peptide. The sequences so combined may be taken from different segments of the primary amino acid sequence of the allergen. For example, a MHC Class II epitope with a high ProPred score can be coupled to a peptide from an IgE-recognized site on the allergen. Preferably, however, those two respective MHC Class II-presented and IgE-recognized sites overlap in the primary sequence of the allergen. Such hybrids combining MHC Class II-presented Ara h2 2 epitopes from Table 2.4 and experimentally determined IgE binding epitopes of Table 2.6 are presented in Table 2.7.
TABLE-US-00011 TABLE 2.6 Experimentally defined IgE-binding Ara h 2 epitopes, which overlap with predicted MHC Class II-presented Ara h 2 epitopes from Table 2.4. PEPTIDE Pos. SEQUENCE Ii-Key SEQ ID NO: 2.6.1 18 HASARQQWEL 10 84 2.6.2 24 QWELQGDRRC 5 85 2.6.3 30 DRRCQSQLER 11 86 2.6.4 42 LRPCEQHLMQ -- 87 Θ 2.6.5 52 KIQRDEDSYE -- 88 2.6.6 130 KRELRNLPQQ -- 89
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined IgE binding epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00012 TABLE 2.7 Hybrids containing predicted MHC Class II Ara h 2 epitopes and overlapping experimentally determined IgE-binding Ara h 2 epitopes. SEQ ID PEPTIDE Pos. Sequence NO: 2.7.1 26 Ac-LRMK-ava-LQGDRRCQS-NH2 90 2.7.2 48 Ac-LRMK-ava-LMQKIQRDE-NH2 91 2.7.3 41 Ac-LRMK-ava-LRPCEQHLM-NH2 92 2.7.4 41/48 Ac-LRMK-ava-LRPCEQHLMOKIQRDE-NH2 93 2.7.5 36 Ac-LRMK-ava-LERANLRPCEQHLMQΘ-NH2 94 2.7.6 12 Ac-LRMK-ava-FLLAAHASARQQWEL-NH2 95
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 2.4 and an IgE binding epitope of Table 2.6. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
[0132]The PEPTIDES of Table 2.7 are characterized as follows. PEPTIDE 2.7.1 contains predicted and experimentally defined MHC Class II epitope PEPTIDE 7.2, which coincides with IgE binding epitope PEPTIDES 2.6.2 and 2.6.3. PEPTIDE 2.7.2 contains predicted and experimentally defined MHC Class II epitope PEPTIDE 7.4, which coincides with IgE binding epitope PEPTIDES 2.6.4 and 2.6.5. PEPTIDE 2.7.3 contains predicted MHC Class II epitope PEPTIDE 7.7, which coincides with IgE binding epitope PEPTIDE 2.6.4. PEPTIDE 2.7.4 contains MHC Class II epitopes PEPTIDE 7.4 and 7.7, and IgE binding epitopes PEPTIDE 2.6.4 and 2.6.5. PEPTIDE 2.7.5 contains MHC Class II epitope PEPTIDE 7.10 overlapping with IgE binding epitope PEPTIDE 2.6.4. PEPTIDE 2.7.6 contains MHC Class II epitope PEPTIDE 7.11, overlapping with IgE binding epitope PEPTIDE 2.6.1.
Example 3
Ii-Key/Ara h 3 Peanut Antigenic Epitope Hybrids
[0133]In another aspect, this invention relates to the design of Ii-Key/Ara h 3 antigenic epitope hybrids. Rabjohn et al. reported the molecular cloning and T cell epitope analysis of the peanut allergen Ara h3 (J Clin Invest. 1999 103:535-42). The sequence of Ara h 3 in Table 3.1 was taken from GenBank gi/3703107/glycin. Predicted MHC Class II epitopes of Ara h 3 are listed in Table 3.2. Ii-Key/Ara h 3 hybrids containing some of the MHC Class II-presented epitopes of Table 3.2 are listed in Table 3.3. Experimentally defined IgE-binding Ara h 3 epitope, overlapping with a predicted MHC Class II-presented Ara h 3 epitope are listed in Table 3.4. Hybrids containing predicted MHC Class II Ara h 3 epitopes overlapping experimentally defined IgE-binding epitopes Table 3.5.
TABLE-US-00013 TABLE 3.1 Deduced amino acid sequence of Ara h 3. 1 rqqpeenacq fqrlnaqrpd nrieseggyi etwnpnnqef ecagvalsrl (SEQ ID NO: 96) 51 vlrrnalrrp fysnapqeif iqqgrgyfgl ifpgcprhye ephtqgrrsq 101 sqrpprrlqg edqsqqqrds hqkvhrfdeg dliavptgva fwlyndhdtd 151 vvavsltdtn nndnqldqfp rrfnlagnte qeflryqqqs rqsrrrslpy 201 spyspqsqpr qeerefsprg qhsrreragq eeeneggnif sgftpefleq 251 afqvddrqiv qnlrgetese eegaivtvrg glrilspdrk rradeeeeyd 301 edeyeydeed rrrgrgsrgr gngieetict asakknigrn rspdiynpqa 351 gslktandln llilrwlgps aeygnlyrna lfvahyntna hsiiyrlrgr 401 ahvqvvdsng nrvydeelqe ghvlvvpqnf avagksqsen feyvafktds 451 rpsianlage nsvidnlpee vvansyglqr eqarqlknnn pfkffvppsq 501 qsprava
TABLE-US-00014 TABLE 3.2 Predicted MHC Class II epitopes of Ara h 3. SEQ ID PEPTIDE Pos. Sequence Score Ii-Key NO: 3.2.1 395 Y R L R G R A H V 6.10 - 97 3.2.2 393 I I Y R L R G R A 4.70 6 98 3.2.3 446 F K T D S R P S I 5.70 -- 99 3.2.4 278 V R G G L R I L S 5.40 -- 100 3.2.5 274 I V T V R G G L R 5.00 10 101 3.2.6 282 L R I L S P D R K 4.70 -- 102 3.2.7 252 F Q V D D R Q I V 5.20 -- 103 3.2.8 364 L R W L G P S A E 5.00 -- 104 3.2.9 362 L I L R W L G P S 4.80 -- 105 3.2.10 173 F N L A G N T E Q 4.80 -- 106 3.2.11 424 L V V P Q N F A V 4.70 -- 107 3.2.12 403 V Q V V D S N G N 4.50 4 108 3.2.13 405 V V D S N G N R V 4.10 6 109 3.2.14 382 F V A H Y N T N A 4.40 -- 110
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00015 TABLE 3.3 Ii-Key/Ara h 3 hybrids containing some of the MHC Class II-presented epitopes of Table 3.2. SEQ ID PEPTIDE Pos. Sequence NO: 3.3.1 393 Ac-LRMK-ava-IIYRLRGRA-NH2 111 3.3.2 395 Ac-LRMK-ava-YRLRGRAHV-NH2 112 3.3.3 393/395 Ac-LRMK-ava-IIYRLRGRAHV-NH2 113 3.3.4 446 Ac-LRMK-ava-FKTDSRPSI-NH2 114 3.3.5 362 Ac-LRMK-ava-LILRWLGPS-NH2 115 3.3.6 364 Ac-LRMK-ava-LRWLGPSAE-NH2 116 3.3.7 362/364 Ac-LRMK-ava-LILRWLGPSAE-NH2 117 3.3.8 252 Ac-LRMK-ava-FQVDDRQIV-NH2 118 3.3.9 173 Ac-LRMK-ava-FNLAGNTEQ-NH2 119 3.3.10 424 Ac-LRMK-ava-LVVPQNFAV-NH2 120 3.3.11 403 Ac-LRMK-ava-VQVVDSNGN-NH2 121 3.3.12 405 Ac-LRMK-ava-VVDSNGNRV-NH2 122 3.3.13 403/405 Ac-LRMK-ava-VQVVDSNGNRV-NH2 123 3.3.14 382 Ac-LRMK-ava-FVAHYNTNA-NH2 124
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 3.2.
[0134]PEPTIDE 3.3.1 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.2. PEPTIDE 3.3.2 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.1. PEPTIDE 3.3.3 is a composite of two ProPred-predicted MHC Class II-presented epitopes (PEPTIDES 3.2.1 and 3.2.2), overlapping by 7 amino acids. PEPTIDE 3.3.4 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.3. PEPTIDE 3.3.5 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.3.6. PEPTIDE 3.2.6 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.8. PEPTIDE 3.3.7 is a composite of two ProPred-predicted MHC Class II-presented epitopes (PEPTIDES 3.2.9 and 3.2.8), overlapping by 7 amino acids. PEPTIDE 3.3.8 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.7. PEPTIDE 3.3.9 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.10. PEPTIDE 3.3.10 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.11. PEPTIDE 3.3.11 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.11. PEPTIDE 3.3.12 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.13. PEPTIDE 3.3.13 is a composite of two ProPred-predicted MHC Class II-presented epitopes (PEPTIDES 3.2.12 and 3.2.13), overlapping by 7 amino acids. PEPTIDE 3.3.14 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.14.
TABLE-US-00016 TABLE 3.4 Experimentally defined IgE-binding Ara h 3 epitope, overlapping with a predicted MHC Class II-presented Ara h 3 epitope. PEPTIDE Pos. Sequence Ii-Key SEQ ID NO: 3.4.1 276 VTVRGGLRILSPDRK 11 125
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00017 TABLE 3.5 Hybrids containing predicted MHC Class II Ara h 3 epitopes overlapping experimentally defined IgE-binding epitopes. SEQ ID PEPTIDE POS. Sequence NO: 3.5.1 274 Ac-LRMK-ava-IVTVRGGLR-NH2 126 3.5.2 277 Ac-LRMK-ava-VRGGLRILS-NH2 127 3.5.3 281 Ac-LRMK-ava-IVTVRGGLRILSPDRK-NH2 128 3.5.4 274/ Ac-LRMK-ava-IVTVRGGLRILSPDRK-NH2 129 277/ 281
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 3.2 and an IgE binding epitope of Table 3.4.
[0135]The PEPTIDES of Table 3.5 are characterized as follows. PEPTIDES 3.5.1-3.5.4 share amino acids between IgE binding and MHC Class II-presented epitopes. PEPTIDE 3.5.1 contains the predicted MHC Class II epitope PEPTIDE 3.2.5, which coincides with experimentally defined IgE binding epitope PEPTIDE 13.1. PEPTIDE 3.5.2 contains the predicted MHC Class II epitope PEPTIDE 3.5.4, which coincides with experimentally defined IgE binding epitope PEPTIDE 3.4.1. PEPTIDE 3.4.3 contains the predicted MHC Class II epitope PEPTIDE 3.2.6, which coincides with experimentally defined IgE binding epitope PEPTIDE 3.4.1. PEPTIDE 3.4.4 is a composite of three ProPred-predicted MHC Class II-presented epitopes (PEPTIDE 3.2.4, 3.2.5 and 3.2.6), which coincide with IgE binding epitope PEPTIDE 3.4.1.
Example 4
Ii-Key/Fel d 1 Cat Dander Antigenic Epitope Hybrids
[0136]In another aspect, this invention relates to the design of Ii-Key/Fel d 1 antigenic epitope hybrids. Such Ii-Key/Fel d 1 antigenic epitope hybrids comprise the Ii-Key motif LRMK (SEQ ID NO: 3) and modifications, joined through a functionally acceptable linker to a MHC Class II-presented epitopes of the Fl d 1 major allergen protein of cat dander. The amino acid sequences of Fel d 1 chains 1 and 2 in Table 4.1 were taken from GenBank gi/1082945/chain 1 and gi/1082946/chain 2 (Morgenstern J P. Proc Natl Acad Sci USA. 1991 88:9640-4). The MHC Class II-presented epitopes of Fel d 1 (chain 1) listed in Table 4.2 include those with the highest scores in the ProPred program analysis. Table 4.3 presents Ii-Key/Fel d 1 (chain 1) hybrids containing some of the predicted MHC Class II-presented epitopes of Table 2. Table 4.4 presents Fel d 1 (chain 1) MHC Class II-presented peptides which elicit allergic responses in cat dander-atopic humans (Haselden B M. J Exp Med. 1999 189: 1885-94). Table 4.5 presents Ii-Key/Fel d 1 (chain 1) hybrids containing some of the experimentally defined MHC Class II-presented epitopes of Table 4.4. Table 4.6 presents predicted MHC Class II-presented epitopes of Fel d 1 (chain 2). Table 4.7 presents designed Ii-Key/Fel d 1 (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.6. Table 4.8 presents Fel d 1 (chain 2) MHC Class II-presented peptides which elicit allergic responses in cat dander-atopic humans (Haselden B M. J Exp Med. 1999 189: 1885-94). Table 4.9. presents designed Ii-Key/Fel d 1 (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.8. Since some of the epitope peptides alone can induce hyporesponsiveness to cat dander allergen challenge in the clinic (Oldfield W L. J Immunol. 2001 167:1734-9; Mazzarella G. Allergy 2000 61:6-9), the corresponding hybrids will be more potent and less susceptible to induce anaphylaxis during clinical testing or therapy. Methods for such analyses and therapy are presented in Larche M. WO99/34826 and U.S. Pat. No. 6,120,769 (2000), which are incorporated herein by reference.
[0137]Asthma is a complex inflammatory disease of the lung characterized by variable airflow obstruction, bronchial hyper-responsiveness, and airway inflammation. Inflammation in asthma consists of airway infiltration by mast cells, lymphocytes, and eosinophils. There is accumulating evidence that CD4+ cells with a Th2-cytokine pattern play a pivotal role in the pathogenesis of asthma. These cells orchestrate the recruitment and activation of the primary effector cells of the allergic response (mast cells and eosinophils), through the release of cytokines such as IL-4, IL-5, and IL-13. Allergic inflammation is also implicated in airway epithelium changes, although the mechanisms by which inflammatory cells and, in particular, T cells interact with the epithelium are not completely clarified.
[0138]Treatment of mice in an ovalbumin-induced asthmatic response with superagonistic Th1-skewing peptide 336E-A (ISQAVHAAHAEINAAGR) (SEQ ID NO: 130) resulted in a Th1-like cytokine profile and a significant decrease in airway eosinophilia and OVA-specific IL-4 and IL-5 production (Janssen E. J Immunol. 2000 164:1580-8; Janssen E M. J Immunol. 2000 165:7207-14). In these studies the wild type sequence (ISQAVHAAHAEINEAGR) (SEQ ID NO: 131) was modified in homologs each with a single alanine substitutions at all non-alanine residue positions.
[0139]In extension of the principles of this study Pene et al. examined the effects of immunotherapy with Fel d 1 peptides on the response to bronchial provocation tests with a standardized Fel d 1 cat extract on Fel d 1-specific serum IgE and IgG levels and in vitro IL-4 and IFN-a production (Pene J. J Allergy Clin Immunol. 1998; 102:571-8). Patients allergic to cats received 6 weekly injections of low dose, medium dose, or high dose of Fel d 1 peptides or a placebo. Six weeks after ending immunotherapy, posttreatment PD20Forced Expiratory Volume/1sec was not significantly different between the treated and placebo groups. However, in the medium- and high-dose groups there was a significant improvement between baseline and posttreatment days. IL-4 release was significantly reduced in the high dose-treated group whereas it was unchanged in the low or medium dose- and in the placebo-treated groups. In all groups, IFN-γ, IgE, and IgG levels remained unchanged. The investigators concluded there was no correlation between the improvement of PD20FEV1 and the decrease in IL-4 production. They suggested that peptide immunotherapy might act by shifting the Fel d 1-induced response of peripheral blood mononuclear cells in vitro from the T.sub.h2-like to the T.sub.h0-like phenotype. The Ii-Key/antigenic epitope hybrids presented below include some of the experimentally tested MHC Class II epitopes of these investigators and can be predicted to be of greater potency and have a wider margin of safety in diagnostic and therapeutic applications, for reasons presented previously.
[0140]The preceding studies followed the preparation of ALLERVAX CAT, a peptide vaccine containing two peptides of 27 amino acids containing regions of multiple MHC Class II-presented epitopes from the Felis domesticus cat allergen Fel d1 chain I were produced FC1P1, LFLTGTPDEYVEQVAQY (SEQ ID NO: 132); FC1P2, EQVAQYKALPVVLENA (SEQ ID NO: 133); and FC1P3, KALP-VVLENARILKNCV (SEQ ID NO: 134).
[0141]The deduced amino acid sequence of Fel d 1 Chain 1 and Chain 2 are presented in Table 4.1 (>gi|1082945|pir∥B56413 major allergen Fel dI chain 1 short form--cat; >gi|1082946|pir∥C56413 major allergen Fel dI chain 2 precursor--cat). Predicted MHC Class-II-presented epitopes of Fel d 1 (chain 1) are listed in Table 4.3. Designed Ii-Key/Fel d 1 (chain 1) hybrids containing some of the predicted MHC Class II-presented epitopes of Table 4.2 are listed in Table 4.3. Experimentally defined Fel d 1 chain 1 MHC Class II-presented epitopes are listed in Table 4.4. Designed Ii-Key/Fel d 1 (chain 1) hybrids containing some of the MHC Class II-presented epitopes of Table 4.4 are listed I Table 4.5. Predicted MHC Class II-presented epitopes of Fel d 1 (chain 2) are listed in Table 4.6. Designed Ii-Key/Fel d (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.6 are listed in Table 4.7. Experimentally determined/Fel d 1 (chain 2) MHC Class II-presented epitopes are listed in Table 4.8. Designed Ii-Key/Fel d 1 (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.8 are listed in Table 4.9.
TABLE-US-00018 TABLE 4.1 Deduced amino acid sequence of Fel d 1. Chain 1. >gi|1082945|pir||B56413 major allergen Fel dI chain 1 short form - cat (SEQ ID NO: 135) 1 mldaalppcp tvaatadcei cpavkrdvdl fltgtpdeyv eqvaqykalp 51 vvlenarilk ncvdakmtee dkenalslld kiytsplc Chain 2. >gi|1082946|pir||C56413 major allergen Fel dI chain 2 precursor - cat (SEQ ID NO: 136) 1 mrgallvlal lvtqalgvkm aetcpifydv ffavangnel lldlsltkvn 51 atepertamk kiqdcyveng lisrvldglv mttissskdc mgeavqntve 101 dlklntlgr
TABLE-US-00019 TABLE 4.2 Predicted MHC ClassII-presented epitopes of Fel d 1 (chain 1). SEQ ID PEPTIDE Pos. Sequence Score Ii-Key NO: 4.2.1 46 Y K A L P V V L E 4.20 -- 137 4.2.2 51 V V L E N A R I L 4.80 -- 138 4.2.3 43 V A Q Y K A L P V 4.70 -- 139 4.2.4 39 Y V E Q V A Q Y K 3.60 8 140 4.2.5 24 V K R D V D L F L 3.4 -- 141 4.2.6 20 I C P A V K R D V 3.1 -- 142 4.2.7 76 L S L L D K I Y T 2.70 8 143 4.2.8 79 L D K I Y T S P L 2.52 11 144 4.2.9 30 L F L T G T P D E 2.40 -- 145
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00020 TABLE 4.3 Designed Ii-Key/Fel d 1 (chain 1) hybrids containing some of the predicted MHC Class II- presented epitopes of Table 4.2. SEQ ID PEPTIDE Pos. Sequence NO: 4.3.1 43 Ac-LRMK-ava-VAQYKALPV-NH2 146 4.3.2 46 Ac-LRMK-ava-YKALPVVLE-NH2 147 4.3.3 51 Ac-LRMK-ava-VVLENARIL-NH2 148 4.3.4 39 Ac-LRMK-ava-YVEQVAQYK-NH2 149 4.3.5 39/43/46/51 Ac-LRMK-ava- 150 YVEQVAQYKALPVVLENARIL-NH2 4.3.6 20 Ac-LRMK-ava-ICPAVKRDV-NH2 151 4.3.7 24 Ac-LRMK-ava-VKRDVDLFL-NH2 152 4.3.8 30 Ac-LRMK-ava-LFLTGTPDE-NH2 153 4.3.9 20/24/30 Ac-LRMK-ava- 154 ICPAVKRDVDLFLTGTPDE-NH2 4.3.10 76 Ac-LRMK-ava-LSLLDKIYT-NH2 155 4.3.11 79 Ac-LRMK-ava-LDKIYTSPL-NH2 156 4.3.12 76/79 Ac-LRMK-ava-LSLLDKIYTSPL- 157 NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00021 TABLE 4.4 Experimentally defined Fel d 1 chain 1 MHC Class II-presented epitopes. PEPTIDE Pos. Sequence Ii-Key SEQ ID NO: 4.4.1 20 EICPAVKRDVDLFLTGT -- 158 4.4.2 30 LFLTGTPDEYVEQVAQY -- 159 4.4.3 41 EQVAQYKALPVVLENA 10 160 4.4.4 47 KALPVVLENARILKNCV -- 161 4.4.5 57 RILKNCVDAKMTEEDKE 5 162 4.4.6 66 KMTEEDKENALSLLDK 2 163 4.4.7 72 KENALSVLDKIYTSPL -- 164
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of certain peptides found to elicit responses in patients with allergy to cat dander (Haselden B M. J Exp Med. 1999 189: 1885-94). Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. PEPTIDE 4.4.2 is from FC1P1; PEPTIDE 4.4.3 is from FC1P2; PEPTIDE 4.4.4 is from FC1P3 (Haselden B M. J Exp Med. 1999 189:1885-94).
TABLE-US-00022 TABLE 4.5 Designed Ii-Key/Fel d 1 (chain 1) hybrids containing some of the MHC Class II-presented epitopes of Table 4.4. SEQ ID PEPTIDE Pos. Sequence NO: 4.5.1 30 Ac-LRMK-ava-LFLTGTPDEYVEQVAQY- 165 NH2 4.5.2 41 Ac-LRMK-ava-EQVAQYKALPVVLENA- 166 NH2 4.5.3 47 Ac-LRMK-ava-KALPVVLENARILKNCV- 167 NH2 4.5.4 57 Ac-LRMK-ava-RILKNCVDAKMTEEDKE- 168 NH2 4.5.5 41/47/57 Ac-LRMK-ava- 169 QVAQYKALPVVLENARILKNCVDAKMTEED KE-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00023 TABLE 4.6 Predicted MHC Class II-presented epitopes of Fel d 1 (chain 2) PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO: 4.6.1 10 LLVTQALGV 6.10 3 170 4.6.2 40 LLLDLSLTK 4.60 -- 171 4.6.3 79 LVMTTISSS 4.37 -- 172 4.6.4 18 VKMAETCPI 4.80 11 173 4.6.5 5 LVLALLVTQ 4.50 -- 174 4.6.6 4 LLVLALLVT 3.90 -- 175
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00024 TABLE 4.7 Designed Ii-Key/Fel d (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.6. SEQ ID PEPTIDE Pos. Sequence NO: 4.7.1 9 Ac-LRMK-ava-LLVTQALGV-NH2 176 4.7.2 5 Ac-LRMK-ava-LVLALLVTQ-NH2 177 4.7.3 4 Ac-LRMK-ava-LLVLALLVT-NH2 178 4.7.4 4/5/9 Ac-LRMK-ava-LLVLALLVTQALGV-NH2 179 4.7.5 17 Ac-LRMK-ava-VKMAETCPI-NH2 180 _4.7.6 39 Ac-LRMK-ava-LLLDLSLTK-NH2 181 4.7.7 78 Ac-LRMK-ava-LVMTTISSS-NH2 182
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope.
TABLE-US-00025 TABLE 4.8 Experimentally determined Fel d 1 (chain 2) MHC Class II-presented epitopes. PEPTIDE Pos. Sequence Ii-Key SEQ ID NO: 4.8.1 46 LTKVNATEPERTAMKK -- 183 4.8.2 57 TAMKKIQDCYVENGLI 6 184 4.8.3 65 CYVENGLISRVLDGLV -- 185 4.8.4 84 ISSSKDCMGEAVQNTV 5 186 4.8.5 94 AVQNTVEDLKLNTLGR -- 187
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00026 TABLE 4.9 Designed Ii-Key/Fel d 1 (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.8. SEQ ID PEPTIDE Pos. Sequence NO: 4.9.1 46 Ac-LRMK-ava-LTKVNATEPERTAMKK-NH2 188 4.9.2 57 Ac-LRMK-ava-TAMKKIQDCYVENGLI-NH2 189 4.9.3 65 Ac-LRMK-ava-CYVENGLISRVLDGLV-NH2 190 4.9.4 84 Ac-LRMK-ava-ISSSKDCMGEAVQNTV-NH2 191 4.9.5 57/65 Ac-LRMK-ava- 192 TAMKKIQDCYVENGLISRVLDGLV-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope.
Example 5
Ii-Key/Phl p 1 Pollen Antigenic Epitope Hybrids
[0142]In another aspect this invention relates to the design and use of Ii-Key/Phl p 1 pollen antigenic epitope hybrids. Laffer and colleagues obtained the cDNA for the major allergen Phl p I from timothy grass (Phleum pratense) and found that the recombinant protein Phl p I inhibits IgE binding to group I allergens prepared form eight different grass species (Laffer S. J Allergy Clin Immunol. 1994 94:689-98). In a study of the T-cell epitopes of Phl p 1, major pollen allergen of timothy grass (Phleum pratense) Schenk S, and colleagues found evidence for crossreacting and non-crossreacting T-cell epitopes within grass group I allergens (Schenk S. J Allergy Clin Immunol. 1995 96:986-96). Immunological characterization of various purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p2, Phl p 5) were characterized with respect to such cross reactions (Vrtala S. J Allergy Clin Immunol. 1996 97:781-7). Various nonanaphylactic synthetic peptides were obtained from antibody-recognized epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination (Focke M. FASEB J. 2001 15:2042-4). Some of these epitopes are incorporated in the Ii-Key/MHC Class II epitope/IgG epitope hybrids of Tables 5.7. In related work, Blaher et al. identified MHC Class II-presented epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen (Blaher B. J Allergy Clin Immunol. 1996 98:124-32).
[0143]The sequence of Phl p I allergen [Phleum pratense] in Table 5.1 was taken from GenBank 473360, Phl p I allergen. Predicted MHC class-II-presented epitopes of Phl p 1 are listed in Table 5.2. Ii-Key/Phl p 1 hybrids containing some of the MHC Class II-presented Phl p 1 epitopes of Table 5.2 are listed in Table 5.3. Experimentally defined MHC Class II-presented epitopes of Phl p 1 are listed in Table 5.4. Ii-Key/Phl p 1 hybrids containing some of the experimentally defined MHC Class II-presented epitopes of Table 5.4 are listed in Table 5.5. Experimentally defined IgE-binding epitopes of Phl p 1 overlapping with MHC Class II-presented Phl p 1 epitopes are listed in Table 5.6. A hybrid peptide including an experimentally defined MHC Class II and IgE binding Php 1 epitope is listed in Table 5.7.
TABLE-US-00027 TABLE 5.1 Deduced amino acid sequence of Phl p 1 pollen protein. (SEQ ID NO: 193) 1 massssvllv vvlfavflgs aygipkvppg pnitatygdk wldakstwyg 51 kptgagpkdn ggacgykdvd kppfsgmtgc gntpifksgr gcgscfeikc 101 tkpeacsgep vvvhitddne epiapyhfdl sghafgamak kgdeqklrsa 151 gelelqfrrv kckypegtkv tfhvekgsnp nylallvkyv ngdgdvvavd 201 ikekgkdkwi elkeswgaiw ridtpdkltg pftvrytteg gtkteaedvi 251 pegwkadtsy esk
TABLE-US-00028 TABLE 5.2 Predicted MHC class II-presented epitopes of Phl P 1. PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO: 5.2.1 0 MASSSSVLL 6.30 -- 194 5.2.2 220 WRIDTPDKL 5.86 5 195 5.2.3 9 VVVLFAVFL 5.80 -- 196 5.2.4 10 VVLFAVLG 6.00 -- 197 5.2.5 6 VLLVVVLFA 5.10 -- 198 5.2.6 96 FEIKCTKPE 5.80 6 199 5.2.7 15 VFLGSAYGI 4.80 -- 200 5.2.8 186 LVKYVNGDG 4.50 9 201 5.2.9 185 LLVKYVNGD 4.50 8 202
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00029 TABLE 5.3 Ii-Key/Phl p 1 hybrids containing some of the MHC Class II-presented Phl p 1 epitopes of Table 5.2. SEQ ID PEPTIDE Pos. Sequence NO: 5.3.1 0 Ac-LRMK-ava-MASSSSVLL-NH2 203 5.3.2 6 Ac-LRMK-ava-VLLVVVLFA-NH2 204 5.3.3 9 Ac-LRMK-ava-VVVLFAVFL-NH2 205 5.3.4 10 Ac-LRMK-ava-VVLFAVFLG-NH2 206 5.3.5 0/6/9/10 Ac-LRMK-ava- 207 MASSSSVLLVVVLFAVFLG-NH2 5.3.6 219 Ac-LRMK-ava-WRIDTPDKL-NH2 208 5.3.7 95 Ac-LRMK-ava-FEIKCTKPE-NH2 209 5.3.8 15 Ac-LRMK-ava-VFLGSAYGI-NH2 210 5.3.9 184 Ac-LRMK-ava-LLVKYVNGD-NH2 211 5.3.10 185 Ac-LRMK-ava-LVKYVNGDG-NH2 212 5.3.11 184/185 Ac-LRMK-ava-LLVKYVNGDG-NH2 213
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope.
TABLE-US-00030 TABLE 5.4 Experimentally defined MHC Class II-presented epitopes of Phl p 1. Ii- SEQ ID PEPTIDE Pos. Sequence Key NO: 5.4.1 96 F E I K C T K P E A C S 6 214 5.4.2 123 I A P Y H F D L S G H A 5 215
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
[0144]The experimentally defined MHC Class II epitopes of Phl p 1, cross react within grass group I allergens--Lol p 1 (ryegrass, Lolium perenne), Sec c 1 (rye, secale cereale) (Schenk S. J Allergy Clin Immunol. 1995 96:986-96). Specifically the epitope of PEPTIDE 5.4.1 cross reacts with Lol p 1 (A97 is replaced by S97) and cross reacts with Sec c 1 (189 is replaced by L89). The epitope of PEPTIDE 5.4.2 cross reacts with Lol p 1 and sec c 1 (A124 is replaced by D124).
TABLE-US-00031 TABLE 5.5 Ii-Key/Phl p 1 hybrids containing some of the experimentally defined MHC Class II-presented epitopes of Table 3. SEQ ID PEPTIDE Pos. Sequence NO: 5.4.1 96 Ac-LRMK-ava-FEIKCTKPEACS-NH2 216 5.4.2 123 Ac-LRMK-ava-IAPYHFDLSGHA-NH2 217
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00032 TABLE 5.6 Experimentally defined IgE-binding epitopes of Phl p 1 overlapping with MHC Class II-presented Phl p 1 epitopes. SEQ ID PEPTIDE Pos. Sequence NO: 5.6.1 24 IPKVPPG PNITATYGDK WLDAKSTWYG KPT 218 5.6.2 65 GYKDVD KPPFSGMTGC GNTPIFKSGR G 219 5.6.3 109 EP VVVHITDDNE EPIAPYHFDL SGHAFGAMA 220 5.6.4 173 HVEKGSNP NYLALLVKYV NGDGDVVAV 221 5.6.5 235 RYTTEG GTKTEAEDVI PEGWKADTSY ESK 222
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. The peptides containing IgE-binding epitopes were defined by Focke and colleagues (Focke M. FASEB J. 2001 15:2042-4). PEPTIDE 5.6.3 includes experimentally defined MHC Class II epitopes of Phl p 1 (Schenk S. J Allergy Clin Immunol. 1995 96:986-96) overlapping with IgE epitope containing peptides (Focke M. FASEB J. 2001 15:2042-4).
TABLE-US-00033 TABLE 5.7 A hybrid peptide including an experimentally defined MHC Class II and IgE binding Php 1 epitope. SEQ ID PEPTIDE Pos. Sequence NO: 5.7.1 109 Ac-LRMK-ava- 223 FEIKCTKPEACSGEPVVVHITDDNE EPIAPYHFDLSGHAFGAMA-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. PEPTIDE 5.7.1 includes Phl p 1 experimentally defined IgE epitopes (Focke M. FASEB J. 2001 15:2042-4) and experimentally defined MHC Class II epitopes of Phl p 1 (Schenk S. J Allergy Clin Immunol. 1995 96:986-96).
Example 6
Ii-Key/Phl p 5a Birch Pollen Antigenic Epitope Hybrids
[0145]In another aspect this invention relates to the design and use of Ii-Key/Phl p 5a birch pollen antigenic epitope hybrids. Multiple T cell epitopes on Bet v I, the major birch pollen allergen, have been determined using specific T cell clones and overlapping peptides (Ebner C. J Immunol. 1993 150:1047-54). Vrtala and colleagues found that the major birch pollen allergen, Bet v 1, can be divided into two fragments each of which contained nonanaphylactic T cell epitopes and are candidates for suppressive immunotherapy (Vrtala S. Int Arch Allergy Immunol. 1997 113:246-8; Vrtala S. J Clin Invest. 1997 99:1673-81). Friedl-Hajek R. and colleagues characterized a highly promiscuous, HLA allele-specific T-cell epitope in the birch major allergen Bet v 1Five Bet v 1-specific T cell clones derived from two birch pollen-allergic individuals and specific for Bet v 1 (Friedl-Hajek R. Clin Exp Allergy. 1999 29:478-87). One of these T cell clones reacted with a Bet v 1 peptide containing amino acid residues 21-33 (BP21), the other two T cell clones reacted with a minimal peptide containing residues 37-45 (BP37). While BP37-specific T cell clones were restricted by a HLA-DQA1*0301/DQB1*0603 heterodimer, BP21 was recognized in a highly promiscuous manner. T cell clones recognizing this sequence were restricted by HLA-DPB1*0201, a HLA-DQA1*0201/DQB1*0201 heterodimer, or HLA-DRB3*0101.
[0146]The sequence of Phl p 5a birch pollen protein in Table 6.1 was taken from GenBank 2851456 (Bufe A. J Allergy Clin Immuno. 1994 94:173-81). Predicted MHC Class II-presented epitopes of Phl p 5a are listed in Table 6.2. Experimentally defined, cross reacting MHC Class II isoepitopes of Phl p 5a and Phl p 5b are listed in Table 6.3 (Muller W. Clin Exp Allergy. 1998 28:1538-48). Designed Ii-Key/antigenic epitope hybrids containing Phl p 5 MHC Class II-presented epitopes are listed in Table 6.4.
TABLE-US-00034 TABLE 6.1 Deduced amino acid sequence of Phl p 5a birch pollen. (SEQ ID NO: 224) 1 adlgygpatp aapaagytpa tpaapagada agkatteeqk liekinagfk 51 aalagagvqp adkyrtfvat fgpasnkafa eglsgepkga aessskaalt 101 skldaaykla yktaegatpe akydayvatl sealriiagt levhavkpaa 151 eevkvipage lqviekvdaa fkvaataana apandkftvf eaafndeika 201 stggayesyk fipaleaavk qayaatvata pevkytvfet alkkaitams 251 eaqkaakpaa aatatataav gaatgaataa tggykv
TABLE-US-00035 TABLE 6.2 Predicted MHC Class II-presented epitopes of Phl p 5a. Ii- SEQ ID PEPTIDE Pos. Sequence Score Key NO: 6.2.1 126 Y V A T L S E A L 8.40 -- 225 6.2.2 153 V K V I P A G E L 5.10 5 226 6.2.3 134 L R I I A G T L E 5.00 -- 227 6.2.4 209 Y K F I P A L E A 4.80 -- 228 6.2.5 206 Y E S Y K F I P A 4.00 -- 229 6.2.6 171 F K V A A T A A N 4.10 2 230 6.2.7 64 Y R T F V A T F G 4.00 -- 231
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00036 TABLE 6.3 Experimentally defined, cross reacting MHC Class II isoepitopes of Phl p 5a and Phl p 5b. Ii- SEQ ID PEPTIDE Pos. Sequence Key NO: 6.3.1 209 Y K F I P A L E A A V K -- 232 6.3.2 161 L Q V I E K V D A A F K 2 233
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. PEPTIDE 6.3.1 corresponds to peptide Phl p 5b(184-195; YKCIPSLEAAVK) (SEQ ID NO: 234) and PEPTIDE 6.3.2 corresponds to peptide Phl p 5b(136-147; LQIIDKIDAAFK (SEQ ID NO: 235) (Muller W. Clin Exp Allergy. 1998 28:1538-48).
TABLE-US-00037 TABLE 6.4 Hybrids containing Phl p 5 MHC Class II- presented epitopes. SEQ ID PEPTIDE Pos. Sequence NO: A. Non-overlapping epitopes. 6.4.1 126 Ac-LRMK-ava-YVATLSEAL-NH2 236 6.4.2 153 Ac-LRMK-ava-VKVIPAGEL-NH2 237 6.4.3 134 Ac-LRMK-ava-LRIIAGTLE-NH2 238 6.4.4 64 Ac-LRMK-ava-YRTFVATFG-NH2 239 B. Overlapping epitopes 6.4.5 209 Ac-LRMK-ava-YKFIPALEA-NH2 240 6.4.6 206 Ac-LRMK-ava-YESYKFIPA-NH2 241 6.4.7 206/209 Ac-LRMK-ava-YESYKFIPALEA-NH2 242 6.4.8 161 Ac-LRMK-ava-LQVIEKVDAAFK-NH2 243 6.4.9 171 Ac-LRMK-ava-FKVAATAAN-NH2 244 6.4.10 161/171 Ac-LRMK-ava- 245 LQVIEKVDAAFKVAATAAN-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
Example 7
Ii-Key/Phospholipase A-2 Bee Venom Antigenic Epitope Hybrids
[0147]In another aspect this invention relates to the design and use of Ii-Key/Phospholipase A-2 bee venom antigenic epitope hybrids. Muller and colleagues successful induced specific T-cell anergy in patients allergic to bee venom with immunotherapy with T-cell recognized peptides of bee venom phospholipase A2 (Muller U. J Allergy Clin Immunol. 1998 101:747-54). Five patients with IgE-mediated systemic allergic reactions to bee stings were treated with a mixture of three T-cell epitope peptides of PLA. Ten patients allergic to BV receiving whole BV immunotherapy served as control subjects. Increasing doses of the peptide mixture, up to a maintenance dose of 100 micrograms, were administered subcutaneously within 2 months. The patients were then challenged with PLA and 1 week later with a bee sting. The cellular and humoral immune response was measured in vitro. No allergic side effects were caused by the peptide immunotherapy, and all patients tolerated the challenge with PLA without systemic allergic symptoms. Two patients developed mild systemic allergic reactions after the bee sting challenge. After peptide immunotherapy, specific proliferative responses to PLA and the peptides in peripheral blood mononuclear cells were decreased in successfully treated patients. The production of TH2 and TH1 cytokines was inhibited, and B cells were not affected in their capacity to produce specific IgE and IgG4 antibodies. Their levels increased after allergen challenge in favor of IgG4. The investigators concluded that immunotherapy of BV allergy with short T-cell peptides of PLA induces epitope-specific anergy in peripheral T cells and changes the specific isotype ratio in a fashion similar to that of conventional immunotherapy in successfully treated patients. Additional MHC Class II-presented candidate epitopes have been identified (Texier C. J Immunol 2000 164:3177-84).
[0148]The sequence of bee venom phospholipase A-2 in Table 7.1 was taken from GenBank 129501 allergen Api m1 (Kuchler K. Eur J Biochem. 1989 184:249-54). Predicted MHC Class II-presented epitopes of the major bee venom allergen phospholipase A-2 are listed in Table 7.2. Table 7.3. Experimentally defined MHC Class II-presented epitopes of the major bee venom allergen phospholipase A-2 are listed in Table 7.3. Ii-Key/PHL A2 hybrids containing some of the MHC Class II-presented PHL A2 epitopes of Table 1 and 2 (nonoverlapping and overlapping epitopes) are listed in Table 7.4.
TABLE-US-00038 TABLE 7.1 Deduced amino acid sequence of Phospholipase A-2 bee venom. (SEQ ID NO: 246) 1 gslfllllst shgwqirdri gdneleerii ypgtlwcghg nkssgpnelg 51 rfkhtdaccr thdmcpdvms ageskhgltn tashtrlscd cddkfydclk 101 nsadtissyf vgkmyfnlid tkcyklehpv tgcgertegr clhytvdksk 151 pkvyqwfdlr ky
TABLE-US-00039 TABLE 7.2 Predicted MHC Class II-presented epitopes of the major bee venom allergen Phospholipase A-2. SEQ ID PEPTIDE Pos. Sequence Score Ii-Key NO: 7.2.1 14 W Q I R D R I G D 7.80 -- 247 7.2.1 4 F L L L L S T S H 5.70 -- 248 7.2.1 110 F V G K M Y F N L 5.50 -- 249 7.2.1 118 L I D T K C Y K L 5.30 -- 250 7.2.1 6 L L L S T S H G W 4.80 -- 251 7.2.1 116 F N L I D T K C Y 4.50 -- 252 7.2.1 5 L L L L S T S H G 4.40 -- 253 7.2.1 52 F K H T D A C C R 4.10 -- 254 7.2.1 124 Y K L E H P V T G 4.08 -- 255
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00040 TABLE 7.3 Experimentally defined MHC Class II-presented epitopes of the major bee venom allergen Phospholipase A-2. Ii- SEQ ID PEPTIDE Pos. Sequence Key NO: 7.3.1 113 K M Y F N L I D T K C Y K -- 256 7.3.2 122 K C Y K L E H P V T G C G 4 257 7.3.3 109 Y F V G K M Y F N L I D T -- 258 7.3.4 141 C L H Y T V D K S K P K 10 259 7.3.5 73 E S K H G L T N T A S H T -- 260 RLSCD
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. The above epitopes were defined by Texier and colleagues and Carballido and colleagues (Texier C. J Immunol. 2000 164:3177-84; Carballido J. J Immunol. 1993 150:3582-91).
TABLE-US-00041 TABLE 7.4 Ii-Key/PHL A2 hybrids containing some of the MHC Class II-presented PHL A2 epitopes of Table 1 and 2. SEQ ID PEPTIDE Pos. Sequence NO: Nonoverlapping epitopes 7.4.1 14 Ac-LRMK-ava-WQIRDRIGD-NH2 261 7.4.2 52 Ac-LRMK-ava-FKHTDACCR-NH2 262 Overlapping epitopes 7.4.3 4 Ac-LRMK-ava-FLLLLSTSH-NH2 263 7.4.4 5 Ac-LRMK-ava-LLLLSTSHG-NH2 264 7.4.5 6 Ac-LRMK-ava-LLLSTSHGW-NH2 265 7.4.6 4/5/6 Ac-LRMK-ava-FLLLLSTSHGW-NH2 266 7.4.7 110 Ac-LRMK-ava-FVGKMYFNL-NH2 267 7.4.8 116 Ac-LRMK-ava-FNLIDTKCY-NH2 268 7.4.9 118 Ac-LRMK-ava-LIDTKCYKL-NH2 269 7.4.10 110/116/ Ac-LRMK-ava-FVGKMYFNLIDTKCYKL- 270 118 NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
Example 8
Bla g 5 glutathione-S-transferase (GST) cockroach antigenic epitope hybrids
[0149]In another aspect this invention relates to the design and use of Ii-Key/Bla g 5 glutathione-S-transferase (GST) cockroach antigenic epitope hybrids. Sensitization to cockroach allergens is associated with the development of asthma. This antigenic protein was found to induce positive skin test reactions in 70% of patients who are allergic to cockroaches. A 23-kDa allergen, glutathione S-transferase (EC 2.5.1.18; GST) was purified from German cockroach (Blattella germanica) by glutathione affinity chromatography (Arruda L K. J Biol Chem. 1997 272:20907-12). The purified protein and recombinant protein had a high level of IgE antibody binding activity. This GST caused positive immediate skin tests in cockroach-allergic patients using as little as 3 pg of recombinant protein. Bla g 1 and has a molecular structure composed of multiple tandem amino-acid repeats. Two consecutive repeats are not identical but form a duplex that constitutes a basic molecular unit of Bla g 1 (Pomes A. Am J Respir Crit Care Med, 2002 165:391-7). The presence of two repeats was not a requirement for IgE antibody binding (Pomes A. Eur J Biochem. 2002 269:3086-3092). That fact supports the view that single linear chains of one repeat in the antigen can be found to express IgE binding epitopes. Bla g 2 is one of the most potent cockroach allergens (prevalence of IgE responses of 60 to 80%) and shows homology to the aspartic proteinase family of enzymes. Mutational destruction of the enzymatic activity did not alter allergenicity (Pomes A. Am J Respir Crit Care Med, 2002 165:391-7). Recombinant proteins of cockroach allergens Bla g 2, Bla g 4 and Bla g 5 and mite group 5 allergens were produced in bacterial expression vectors and demonstrated strong immediate skin and serum IgE antibody responses in cockroach-allergic patients (Chapman M D. Int Arch Allergy Immunol. 1997 113:102-4). Each of the recombinant allergens retained biologic activity and the investigators suggested that cocktails of two to four recombinant allergens could be used for diagnostic or therapeutic purposes. No dominant linear IgE-binding epitopes have been reported, but when they become determined, Ii-Key/Bla g MHC Class II epitope/BLA g IgE-recognized hybrid peptides can be designed and tested.
[0150]The sequence of cockroach allergen BLA g 5 in Table 8.1 was taken from GenBank 6225491 (Arruda L K. J Biol Chem. 1997 272:20907-12). Predicted MHC Class II epitopes of cockroach allergen Bla g 5 are presented in Table 8.2. Experimentally defined MHC Class II epitopes of Bla g 5 (Papouchado B G. Tissue Antigens. 2000 55:303-11) are presented in Table 8.3.
TABLE-US-00042 TABLE 8.1 Deduced amino acid sequence of cockroach allergen Bla g 5. (SEQ ID NO: 271) 1 mapsykltyc pvkalgepir fllsygekdf edyrfqegdw pnlkpsmpfg 51 ktpvleidgk qthqsvaisr ylgkqfglsg kddwenleid mivdtisdfr 151 aaianyhyda denskqkkwd plkketipyy tkkfdevvka nggylaagkl 201 twadfyfvai ldylnhmake dlvanqpnlk alrekvlglp aikawvakrp 251 ptdl
TABLE-US-00043 TABLE 8.2 Predicted MHC Class II-presented epitopes of cockroach allergen Bla g 5. PEPTIDE Sequence Ii- SEQ ID NO: Pos. 1 2 3 4 5 6 7 8 9 Score Key NO: 8.2.1 49 F G K T P V L E I 7.60 -- 272 8.2.2 245 W V A K R P P T D 5.85 -- 273 8.2.3 91 M I V D T I S D F 5.57 -- 274 8.2.4 205 F Y F V A I L D Y 5.40 -- 275 8.2.5 19 I R F L L S Y G E 5.10 2 276 8.2.6 55 L E I D G K Q T H 4.60 7 277 8.2.7 99 F R A A I A N Y H 4.20 -- 278
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00044 TABLE 8.3 Experimentally defined MHC Class II epitopes of Bla g 5. PEPTIDE SEQ ID NO: Pos. Sequence Ii-Key NO: 8.3.1 92 IVDTISDFRAAIANYHYDAD -- 279 8.3.1 212 DYLNHMAKEDLVANQPNLKA -- 280
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
Example 9
Ii-Key/CEA Antigenic Epitope Hybrids
[0151]Carcinoembryonic antigen (CEA) is a tumor associated antigen (TAA) that is expressed in tumors including colon, breast and pancreas. The protein and the cDNA have been used for therapeutic tumor vaccines. A recombinant vaccinia-CEA vaccine has been used to generation cytotoxic T cells specific for human carcinoembryonic antigen epitopes (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Ii-Key hybrids can be used to develop T helper cell responses to this tumor-associated antigen prior to DNA vaccines of any form. Thus, the clinical value of such a recombinant vaccinia-CEA construct can be enhanced substantially with the products and methods of this invention, as described in this disclosure. Additional CEA vaccination procedures, in which Ii-Key/CEA antigenic epitope hybrids can be applied, are presented below.
[0152]Reisfeld and colleagues demonstrated that an oral DNA vaccine against human CEA prevented growth and dissemination of Lewis lung carcinoma in CEA transgenic mice (Niethammer A G. Vaccine 2001 20:421-9). A DNA vaccine encoding human CEA broke peripheral T-cell tolerance toward this antigen expressed by Lewis lung carcinoma stably transduced with CEA in C57BL/6J mice transgenic for CEA. The vaccine was delivered by oral gavage with an attenuated strain of Salmonella typhimurium (SL7207), and boosted with an antibody-IL2 fusion protein. Both CTL and antigen-presenting dendritic cells were activated as indicated by a decisive increase in their respective activation markers CD2, CD25, CD28 as well as CD48 and CD80.
[0153]Stevenson and colleagues demonstrated that DNA fusion vaccine including MHC Class II epitopes of tetanus toxoid along with a tumor antigen of interest (here CEA) induced cytotoxic T cell responses against defined peptides. Fusion of the fragment C of tetanus toxin to a CEA sequence promoted antibody and CD4+ T cell responses against tested B cell tumors. Using only the first domain of tetanus toxoid, which contains a "universal" helper epitope, followed by two known CTL-recognized epitopes of CEA, they found strong CTL responses to each CTL-recognized peptide to be induced by the engineered construct.
[0154]Diagnostic assays with Ii-Key/antigenic epitope hybrids can be used to monitor therapy and predict outcomes in patients with CEA-positive tumors, as indicated from the following study. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses to the same CEA peptide and to an influenza (Flu) control peptide (Arlen P. Cancer Immunol Immunother. 2000 49:517-29). The first trial consisted of three monthly vaccinations of CEA peptide (designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant (designated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations with avipox-CEA (designated VAA). Few, if any, CEA-specific T cell responses were seen in patients receiving PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no increases in Flu-specific responses. CEA-specific IgG responses developed in patients following recombinant CEA poxvirus vaccinations. T cell responses to the CEA peptide were significantly increased after immunization with the recombinant poxvirus vaccine, as compared with the peptide vaccine (p=0.028). Clearly poxvirus recombinant-based vaccines are more potent in initiating tumor-antigen-specific T cell responses than are peptide vaccines. Their activity can be further enhanced, by prior vaccination with Ii-Key/CEA antigenic epitope hybrids.
[0155]In the case of tumor antigens such as CEA, Ii-Key/MHC Class II-presented antigenic epitope hybrids create T helper cell responses that augment the development of immune responses to CEA MHC Class I epitopes, for example through dendritic cell licensing. Such CTL activation by MHC Class I epitopes can be generated also by incorporating such MHC Class I epitopes in an Ii-Key MHC Class II-presented hybrids. Several MHC Class I epitope of CEA have been experimentally determined (Kawashima I. Hum Immunol. 1998 59:1-14; Nukaya I. Int J Cancer. 1999 80:92-7; Table 5). Such peptides have been discovered by various techniques. Nested deletions of the cDNA for the antigen of interest lead to protein products, which can be assayed for stimulation of CD8+ cells lines, which recognize the antigen. Given various cell lines recognizing individual epitopes, the localization of T cell epitopes can be approximated within the primary sequence, by analyzing the reactions of each T cell clone to the nested deletion cDNA constructs. Then a library of overlapping peptides through biologically active target regions can be assayed to define exactly the individual determinants. The binding of such peptides to immunopurified MHC Class I molecules can also be assayed, for example by inhibition of binding of a radiolabeled standard peptide to MHC molecules (Kawashima I. Hum Immunol. 1998 59:1-14). The MHC Class I molecules can be immunopurified and bound into microtiter plates, in which the various components of the assay are added sequentially, with appropriate washings. Alternatively the MHC Class I molecules can be detergent-solubilized without purification, for example from a microsomal membrane preparation of a cultured lymphoblastoid cell line, and the complexes separated in a gel filtration column, with the bound radioactive peptide being separated in the protein complexes from the unbound, free peptide. In the work of Kawashima, initial studies were performed with HLA-A2.1 molecules. The highly reactive peptide 9.5.2, which induced vigorous anti-tumor CTL responses, also bound tightly to other common HLA alleles of the A2 supertype (A2.2, A2.3, A2.6 and A6802), thus demonstrating a potential in providing broad and not ethnically biased population coverage. CTL lines were used to identify peptides 9.5.4 and 9.5.6, which elicited CTL lines that lysed tumor cells expressing HLA-A24 and CEA. The cytotoxicity to tumor cells by the CTL lines was antigen-specific since it was inhibited by peptide-pulsed cold target cells as well as by monoclonal antibodies to MHC Class I and CD3 molecules. Similar methods can be used to characterize the biological responses induced by Ii-Key/MHC Class II epitope/MHC Class I epitope hybrids of this disclosure.
[0156]Alternatively, such peptides are identified with algorithms for the prediction of MHC Class I and Class II T cell-recognized epitopes (Lu J. Cancer Res. 2000 60:5223-7). These computer-based predictive algorithms, which are available on the Internet (Parker K C. J Immunol. 1992 149:3580-7; Rammensee H G. Immunogenetics. 1995 41:178-228), were used to identify HL4-B7-restricted CTL epitopes for carcinoembryonic antigen (CEA). Of three candidate peptides, CEA9(632) (IPQQHTQVL) (SEQ ID NO: 281) induced primary CTL responses in lymphocytes from HLA-B7+ normal blood donors when dendritic cells were used as antigen-presenting cells. These CTLs were efficient in killing tumor cells that expressed HLA-B7 and produced CEA.
[0157]Cell lines reflecting the natural T-cell response against MHC Class I epitopes of epithelial cell adhesion molecule, Her-2/neu, and carcinoembryonic antigen in patients with colorectal cancer have been used to identify the respective antigenic epitopes (Nagorsen D. Cancer Res. 2000 60:4850-4). Antigens of epithelial cell adhesion molecule (Ep-CAM), her-2/neu, and CEA were potential targets in antigen-specific vaccination-based cancer therapy. The investigators tested whether a natural specific T-cell response against these antigens already exists in patients with colorectal carcinoma. The IFN-gamma ELISPOT assay was used to detect circulating TAA-reactive T cells directly ex vivo in unstimulated peripheral blood mononuclear cells. They determined that seven of 22 patients, but none of the 8 healthy subjects, had T cells specifically secreting IFN-gamma in response to antigen peptides (n=4, Ep-CAM; n=5, her-2/neu; n=6, CEA). T-cell responses occurred only in patients with metastatic disease (Dukes' stages C and D). The results of this study indicate that natural T-cell responses against tumor antigens occur in approximately one-half of colorectal carcinoma patients with involvement of lymph nodes or distant metastases, but not in colorectal carcinoma patients with disease confined to the bowel tract. Ii-Key/antigenic epitope hybrids containing MHC Class II epitopes can be used to vaccinate patients with localized and metastatic disease against their tumors.
[0158]The amino acid sequence of CEA was obtained from GenBank as 11386171 carcinoembryonic antigen-related cell adhesion molecule 5; carcinoembryonic antigen [Homo sapiens] (Table 1). The primary sequence of human carcinoembryonic antigen (CEA) was deduced from cDNA sequence (Oikawa S. Biochem Biophys Res Commun. 1987 142:511-8). The carcinoembryonic antigen (CEA) contains multiple immunoglobulin-like domains (Oikawa S. Biochem Biophys Res Commun. 1987 144:634-42). Predicted MHC Class II-presented epitopes of CEA are listed in Table 9.2. Designed Ii-Key/CEA hybrids containing some of the MHC Class II-presented epitopes of CEA in Table 9.2 are listed in Table 9.3. Predicted MHC Class I-presented CEA epitopes are listed in Table 9.4. Experimentally defined MHC Class I epitopes of CEA are listed in Table 9.5. Ii-Key/MHC Class II/MHC Class I CEA hybrids are listed in Table 9.6.
TABLE-US-00045 TABLE 9.1 Deduced amino acid sequence of CEA. 1 mespsapphr wcipwqrlll taslltfwnp pttaklties tpfnvaegke (SEQ ID NO: 282) 51 vlllvhnlpq hlfgyswykg ervdgnrqii gyvigtqqat pgpaysgrei 101 iypnaslliq niiqndtgfy tlhviksdlv neeatgqfrv ypelpkpsis 151 snnskpvedk davaftcepe tqdatylwwv nnqslpvspr lqlsngnrtl 201 tlfnvtrndt asykcetqnp vsarrsdsvi lnvlygpdap tisplntsyr 251 sgenlnlsch aasnppaqys wfvngtfqqs tqelfipnit vnnsgsytcq 301 ahnsdtglnr ttvttitvya eppkpfitsn nsnpvededa valtcepeiq 351 nttylwwvnn qslpvsprlq lsndnrtltl lsvtrndvgp yecgiqnels 401 vdhsdpviln vlygpddpti spsytyyrpg vnlslschaa snppaqyswl 451 idgniqqhtq elfisnitek nsglytcqan nsasghsrtt vktitvsael 501 pkpsissnns kpvedkdava ftcepeaqnt tylwwvngqs lpvsprlqls 551 ngnrtltlfn vtrndarayv cgiqnsvsan rsdpvtldvl ygpdtpiisp 601 pdssylsgan lnlschsasn pspqyswrin gipqqhtqvl fiakitpnnn 651 gtyacfvsnl atgrnnsivk sitvsasgts pglsagatvg imigvlvgva 701 li
TABLE-US-00046 TABLE 9.2 Predicted MHC Class II-presented epitopes of CEA. PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO: 9.2.1 427 YRPGVNLSL 6.40 -- 283 9.2.2 535 WVNGQSLPV 6.30 -- 284 9.2.3 179 WVNNQSLPV 5.50 -- 285 357 6.30 9.2.4 627 WRINGIPQQ 5.80 -- 286 9.2.5 249 YRSGENLNL 5.50 -- 287 9.2.6 52 LLLVHNLPQ 5.40 -- 288 9.2.7 449 WLIDGNIQQ 5.10 12 289 5.78 9.2.8 591 YGPDTPIIS 5.10 -- 290 9.2.9 119 FYTLHVIK S 5.10 -- 291 9.2.10 79 IIGYVIGTQ 5.00 -- 292
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00047 TABLE 9.3 Designed Ii-Key/CEA hybrids containing same of the MHC Class II-presented epitopes of CEA in Table 9.2. A. Non-overlapping epitopes PEPTIDE Pos. Sequence SEQ ID NO: 9.3.1 179 Ac-LRMK-ava-WVNNQSLPV-NH2 293 357 535 9.3.2 427 Ac-LRMK-ava-YRPGVNLSL-NH2 294 9.3.3 627 Ac-LRMK-ava-WRINGIPQQ-NH2 295 9.3.4 249 Ac-LRMK-ava-YRSGENLNL-NH2 296
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 9.2. CEA contains seven extracellular domains, which are strikingly homologous to each other. This fact explains the repeated identical epitopes that starts at positions 178, 356, and 534 in this Table (Oikawa S. Biochem Biophys Res Commun. 187 144:634-42).
TABLE-US-00048 TABLE 9.4 Predicted MHC Class I-presented CEA epitopes. PEPTIDE Pos. Sequence Score SEQ ID NO: 9.4.1 61 HLFGYSWYK 1350 297 9.4.2 425 TYYRPGVNL 200 298 9.4.3 652 TYACFVSNL 200 299 9.4.4 691 IMIGVLVGV 196 300 9.4.5 605 YLSGANLNL 98 301
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptide 9.3.1 are presented by HLA-A3. Peptides 9.3.2 and 9.3.2 are presented by HLA-A24. Peptides 9.3.4 and 9.3.5 are presented by HLA-A2.1. The MHC Class I-presented epitopes of this Table were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/).
TABLE-US-00049 TABLE 9.5 Experimentally defined MHC Class I epitopes of CEA. PEPTIDE Pos. Sequence SEQ ID NO: 9.5.1 691 IMIGVLVGV 302 9.5.2 24 LMTFWNPPV 303 9.5.3 605 YLSGANLNL 304 9.5.4 268 QYSWFVNGTF 305 9.5.5 652 TYACFVSNL 306 9.5.6 61 HLFYSWYK 307
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class I-presented epitope. Peptides 9.5.1, 9.5.2, 9.5.3 are presented by HLA-A2.1 and 9.5.6 is presented by HLA-A3 (Kawashima I. Hum Immunol. 1998 59:1-14). Peptides 9.5.4 and 9.5.5 are presented by HLA-A24 (Nukaya I. Int J Cancer. 1999 80:92-7). Peptide 9.5.2 is presented by HLA-A2.1 and it and LLTFWNPPV (SEQ ID NO: 308) are engineered CEA epitopes with respect to the wild type sequence LLTFWNPPT (SEQ ID NO: 309).
TABLE-US-00050 TABLE 9.6 Ii-Key/MHC Class II/MHC Class I CEA hybrids. SEQ ID PEPTIDE Pos. Sequence NO: 9.6.1 II: 179, 357, Ac-LRMK-ava-WVNNQSLPV- 310 535, IMIGVLVGV-NH2 I: 691 9.6.2 II: 427, Ac-LRMK-ava-TYYRPGVNLSL- 311 I: 425 NH2 9.6.3 II: 249, Ac-LRMK-ava-YRSGENLNL- 312 I: 268 QYSWFVNGTF-NH2 9.6.4 II: 52, Ac-LRMK-ava-LLLVHNLPQ- 313 I: 61 HLFYSWYK-NH2
Ii-Key/MHC Class II/MHC Class I CEA hybrids. The sequence position of the MHC Class II epitope is indicated: II:residue position of first epitope amino acid, and of the MHC Class I epitope is indicated: I:residue position of first epitope amino acid.
Example 10
Ii-Key/Ca-125 Cancer Antigenic Epitope Hybrids
[0159]The ovarian cancer antigen CA-125 is used in immunotherapeutic vaccinations. In one case, vaccination with a mixed vaccine of autogenous and allogeneic breast cancer cells and tumor associated antigens including the breast cancer antigen CA15.3, the carcinoembryonic antigen (CEA) and the ovarian cancer antigen CA125, resulted in immune and clinical responses in breast cancer patients (Jiang X P. Cancer Biother Radiopharm. 2000 15:495-505). The vaccine induced a significant increase in post-vaccination lymphocyte proliferative responses to AUTOC, CA15.3, CEA and CA125 but not ALLOC, compared to pre-vaccination (p<0.05, p<0.01, p<0.05, p<0.01 and p>0.05, respectively, a paired t Test).
[0160]The amino acid sequence of CA125 ovarian cancer antigen mucin 16 [Homo sa . . . [gi:14971110] as listed in Genebank is presented in Table 10.1. Predicted MHC Class II-presented epitopes of CA125, ovarian cancer antigen are listed in Table 10.2. Ii-Key/CA 125 hybrids containing some of the MHC Class II-presented epitopes of Table 10.2 are listed in Table 10.3. Predicted MHC Class I-presented epitopes of CA 125 are listed in Table 10.4. Ii-Key/MHC II epitope/MHC I epitope hybrids are listed in Table 10.5.
TABLE-US-00051 TABLE 10.1 Deduced amino acid sequence of CA125 ovarian cancer antigen (SEQ ID NO: 314) 1 rvdpigpgld rerlywelsq ltnsitelgp ytldrdslyv ngfnpwssvp 51 ttstpgtstv hlatsgtpss lpghtapvpl lipftlnfti tnlhyeenmq 101 hpgsrkfntt ervlqgllkp lfkstsvgpl ysgcrltllr pekhgaatgv 151 daictlrldp tgpgldrerl ywelsqltns vtelgpytld rdslyvngft 201 hrssvpttsi pgtsavhlet sgtpaslpgh tapgpllvpf tlnftitnlq 251 yeedmrhpgs rkfnttervl qgllkplfks tsvgplysgc rltllrpekr 301 gaatgvdtic thrldplnpg ldreqlywel skltrgiiel gpylldrgsl 351 yvngfthrnf vpitstpgts tvhlgtsetp sslprpivpg pllvpftlnf 401 titnlqyeea mrhpgsrkfn ttervlqgll rplfkntsig plysscrltl 451 lrpekdkaat rvdaicthhp dpqspglnre qlywelsqlt hgitelgpyt 501 ldrdslyvdg fthwspiptt stpgtsivnl gtsgippslp ettatgpllv 551 pftlnftitn lqyeenmghp gsrkfnites vlqgllkplf kstsvgplys 601 gcrltllrpe kdgvatrvda icthrpdpki pgldrqqlyw elsqlthsit 651 elgpytldrd slyvngftqr ssvpttstpg tftvqpetse tpsslpgpta 701 tgpvllpftl nftiinlqye edmhrpgsrk fnttervlqg llmplfknts 751 vsslysgcrl tllrpekdga atrvdavcth rpdpkspgld rerlywklsq 801 lthgitelgp ytldrhslyv ngfthqssmt ttrtpdtstm hlatsrtpas 851 lsgpttaspl lvlftinfti tnlryeenmh hpgsrkfntt ervlqgllrp 901 vfkntsvgpl ysgcrltllr pkkdgaatkv daictyrpdp kspgldreql 951 ywelsqlths itelgpytld rdslyvngft qrssvpttsi pgtptvdlgt 1001 sgtpvskpgp saaspllvlf tlnftitnlr yeenmqhpgs rkfnttervl 1051 qgllrslfks tsvgplysgc rltllrpekd gtatgvdaic thhpdpkspr 1101 ldreqlywel sqlthnitel gpyaldndsl fvngfthrss vsttstpgtp 1151 tvylgasktp asifgpsaas hllilftlnf titnlryeen mwpgsrkfnt 1201 tervlqgllr plfkntsvgp lysgcrltll rpekdgeatg vdaicthrpd 1251 ptgpgldreq lylelsqlth sitelgpytl drdslyvngf thrssvptts 1301 tgvvseepft lnftinnlry madmgqpgsl kfnitdnvmq hllsplfqrs 1351 slgarytgcr vialrsvkng aetrvdllct ylqplsgpgl pikqvfhels 1401 qqthgitrlg pysldkdsly lngynepgpd eppttpkpat tflpplseat 1451 tamgyhlktl tlnftisnlq yspdmgkgsa tfnstegvlq hllrplfqks 1501 smgpfylgcq lislrpekdg aatgvdttct yhpdpvgpgl diqqlywels 1551 qlthgvtqlg fyvldrdslf ingyapqnls irgeyqinfh ivnwnlsnpd 1601 ptsseyitll rdiqdkvttl ykgsqlhdtf rfclvtnltm dsvlvtvkal 1651 fssnldpslv eqvfldktln asfhwlgsty qlvdihvtem essvyqptss 1701 sstqhfypnf titnlpysqd kaqpgttnyq rnkrniedal nqlfrnssik 1751 syfsdcqvst frsvpnrhht gvdslcnfsp larrvdrvai yeeflrmtrn 1801 gtqlqnftld rssvlvdgys pnrnepltgn sdlpfwavil iglagllgli 1851 tclicgvlvt trrrkkegey nvqqqcpgyy qshldledlq
TABLE-US-00052 TABLE 10.2 Predicted MHC Class II-presented epitopes of CA125, ovarian cancer antigen. PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO: 10.2.1 1630 FRFCLVTNL 7.40 9 315 10.2.2 1018 VLFTLNFTI 7.10 -- 316 10.2.3 1174 ILFTLNFTI 7.10 -- 317 10.2.4 156 LRLDPTGPG 6.80 -- 318 10.2.5 1017 LVLFTLNFT 6.20 -- 319 10.2.6 861 LVLFTINFT 6.05 -- 320 10.2.7 527 IVNLGTSGI 5.70 -- 321 10.2.8 1318 LRYMADMGQ 5.70 -- 322 10.2.9 1029 LRYEENMQH 5.68 -- 323 10.2.10 873 LRYEENMHH 5.68 -- 324 10.2.11 1663 VFLDKTLNA 5.60 10 325 10.2.12 1172 LLILFTLNF 5.60 -- 326 10.2.13 936 YRPDPKSPG 5.57 2 327 10.2.14 393 LVPFTLNFT 5.50 3 328 10.2.15 430 LRPLFKNTS 5.40 -- 329 10.2.16 1185 LRYEENMWP 5.30 9 330 10.2.17 1634 LVTNLTMDS 5.30 -- 331 10.2.18 360 FVPITSTPG 5.20 11 332 10.2.19 1209 LRPLFKNTS 5.10 -- 333 10.2.20 898 LRPVFKNTS 5.10 -- 334 10.2.21 1531 YHPDPVGPG 5.10 -- 335
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Because CA 125/MUC 16 is characterized by nine partially conserved tandem repeats (156 amino acids each) in an N-terminal region, similar predicted epitopes have different starting positions (e.g., start position 1017, 1173, 860, or 897, 1208, or 1184, 872, 1028).
TABLE-US-00053 TABLE 10.3 Ii-Key/CA125 hybrids containing some of the MHC Class II-presented epitopes of Table 10.2. SEQ ID PEPTIDE Pos. Sequence NO: A. Conserved tandem-repeats epitopes 10.3.1 1017 Ac-LRMK-ava-VLFTLNFTI-NH2 336 10.3.2 1173 Ac-LRMK-ava-ILFTLNFTI-NH2 337 10.3.3 860 Ac-LRMK-ava-LVLFTINFT-NH2 338 10.3.4 1028 Ac-LRMK-ava-LRYEENMQH-NH2 339 10.3.5 872 Ac-LRMK-ava-LRYEENMHH-NH2 340 10.3.6 1184 Ac-LRMK-ava-LRYEENMWP-NH2 341 B. Overlapping MHC II epitopes 10.3.7 1629 Ac-LRMK-ava-FRFCLVTNL-NH2 342 10.3.8 1633 Ac-LRMK-ava-LVTNLTMDS-NH2 343 10.3.9 1629/ Ac-LRMK-ava- 344 1633 FRFCLVTNLTMDS-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 10.2.
TABLE-US-00054 TABLE 10.4 Predicted MHC Class I-presented epitopes of CA 125. PEPTIDE Pos. Sequence Score SEQ ID NO: 10.4.1 1675 WLGSTYQLV 478 345 10.4.2 1018 VLFTLNFTI 381 346 10.4.3 1174 ILFTLNFTI 381 347 10.4.4 862 VLFTINFTI 381 348 10.4.5 344 LLDRGSLYV 260 349 10.4.6 1506 YLGCQLISL 226 350 10.4.7 1668 TLNASFHWL 223 351 10.4.8 1555 GVTQLGFYV 194 352 10.4.9 1845 GLLGLITCL 182 353 10.4.10 32 KLTRGIIEL 172 354
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted HLA-A2.1-resented epitope. Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). The MHC Class I-presented epitopes of this Table were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/).
TABLE-US-00055 TABLE 10.5 Ii-Key/MHC II epitope/MHC I epitope hybrids. Ii-Key/MHC Class SEQ ID PEPTIDE Pos. Sequence NO: 10.5.1 1630 Ac-LRMK-ava-FRFCLVTNL- 355 NH2 10.5.2 II: 392 Ac-LRMK-ava-LVPFTLNFTI- 356 I: 394, 238, NH2 82, 550
Ii-Key/MHC Class II/MHC Class I CEA hybrids. The sequence position of the MHC Class II epitope is indicated: II:residue position of first epitope amino acid, and of the MHC Class I epitope is indicated: I:residue position of first epitope amino acid. In peptide 10.5.1 the MHC Class II-predicted and the MHC Class I-predicted epitopes overlap precisely. In peptide 10.5.2 an MHC Class-predicted epitope starting at residue position 392 overlaps with the sequence of a MHC Class I-predicted epitope which starts (and is repeated at) residue positions 394, 238, 82, 550.
Example 11
Ii-Key/PSA Antigenic Epitope Hybrids
[0161]The identification of T cell specific epitopes within the coding sequence of PSA has led to the development of various vaccine strategies that target PSA in an attempt to treat established prostate cancer (Kaufman H L. Expert Opin Biol Ther. 2002 2:395-408). These strategies have included HLA-restricted PSA peptides, dendritic cells pulsed with PSA, recombinant viruses expressing PSA and combinations with different cytokines and cell interaction molecules. Many of these methods are enhanced by use of the products and methods of this disclosure.
[0162]PSA-recombinant pox vaccine constructs are immunogenic and induce antibody responses to a multitude of surface antigens on prostate tumor cell lines by epitope or determinant spreading after stimulation of the immune system by PSA immunization (Cavacini L A. Clin Cancer Res. 2002 8:368-73). Determinant spreading in the antibody responses to prostate cell surface antigens was observed in patients immunized with prostate-specific antigen encoded by recombinant pox vectors. The serum IgG response to cell surface antigens expressed on LNCAP (PSA-positive) and PC-3 (PSA-negative) prostate cancer cell lines were analyzed in individuals with advanced disease receiving vaccinia- or fowlpox-expressed PSA (v-PSA or f-PSA, respectively). Sera from all seven patients in a Phase I study of v-PSA, collected prior to the third immunization, reacted with both prostate tumor cell lines. The majority of individuals (n=12) in a Phase II trial of v-PSA and f-PSA developed sustainable antibody responses to cell surface antigens on the prostate tumor cell lines. The magnitude and kinetics of these responses depended on the immunization schedule.
[0163]Whiteside and colleagues demonstrated recovery of zeta-chain expression and changes in spontaneous IL-10 production after PSA-based vaccines in patients with prostate cancer (Meidenbauer N. Br J Cancer. 2002 86:168-78). In order to determine a mechanism by which circulating T lymphocytes of patients with prostate cancer have been reported to have functional deficits, including low or absent zeta-chain expression, 10 patients treated with recombinant human prostate specific antigen plus GM-CSF and eight others receiving PSA plus oil emulsion were evaluated. Prior to therapy, the patients had significantly lower zeta-chain expression in circulating CD3+ cells and a higher percentage of zeta-chain negative CD3+ and CD4+ cells than normal donors. The patients' peripheral blood mononuclear cells spontaneously produced more IL-10 ex vivo than those of normal controls. After vaccination, recovery of zeta-chain expression was observed in 50% of patients in both clinical trials. Also, spontaneous IL-10 secretion by peripheral blood mononuclear cells decreased following immunotherapy in patients treated with PSA and GM-CSF. Such therapies will be greatly augmented by products and methods of this disclosure.
[0164]Mann and colleagues demonstrated enhanced CD4+ and CD8+ T cell responses after exposure to PSA alone, PSA targeted to the mannose receptor (mannosylated PSA (PSA-m)), or PSA targeted to Fc receptors by combining PSA with an anti-PSA antibody (AR47.47) (Berlyn K A. Clin Immunol. 2001 101:276-83). PSA and PSA-m are processed primarily through pathways that favor MHC Class II presentation, while the PSA/anti-PSA immune complexes are processed through both Class I and Class II pathways in monocyte-derived dendritic cells.
[0165]Gilboa and colleagues demonstrated that autologous dendritic cells transfected with PSA RNA stimulate CTL responses against metastatic prostate tumors (Heiser A. J Clin Invest. 2002 109:409-17). Autologous dendritic cells transfected with mRNA encoding prostate-specific antigen (PSA) stimulate potent, T cell-mediated antitumor immune responses in vitro. A phase I trial evaluated this strategy for safety, feasibility, and efficacy to induce T cell responses against the PSA in patients with metastatic prostate cancer. In 13 subjects, escalating doses of PSA mRNA-transfected dendritic cells were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope of serum PSA levels in six of seven subjects.
[0166]Schlom and colleagues characterized an agonist epitope designated PSA-3A ("A" for agonist) of the PSA-3 CTL epitope which demonstrated enhanced binding to the HLA-A2 allele and enhanced stability of the peptide-MHC complex (Terasawa H. Clin Cancer Res. 2002 8:41-53). T-cell lines generated with either the PSA-3 or the PSA-3A peptide showed higher levels of lysis of targets pulsed with the PSA-3A peptide than those targets pulsed with the PSA-3 peptide. T cells stimulated with dendritic cells (dendritic cells) pulsed with PSA-3A peptide produced higher levels of IFN-gamma than did dendritic cells pulsed with PSA-3 peptide. Dendritic cells infected with a recombinant vaccinia virus containing the agonist amino acid change within the entire PSA gene (designated rV-PSA-3A) were more effective than dendritic cells infected with the rV-PSA vector in enhancing IFN-gamma production by T cells. Finally, the PSA-3A agonist was shown to induce higher levels of T-cell activation, compared with the PSA-3 peptide, in an in vivo model using HLA-A2.1/K(b) transgenic mice. These studies thus demonstrated the potential use of the PSA-3A agonist epitope in both peptide- and vector-mediated immunotherapy protocols for prostate cancer. Such results can be bettered with the products and methods of this disclosure.
[0167]Recombinant PSA proteins incorporating 6×His (SEQ ID NO: 357) residues were synthesized for magnetic bead attachment allowing antigen isolation and delivery to APC for processing and presentation (Turner M J. J Immunol Meth. 1998 256:107-19). PSA deletion constructs were generated by amplifying 3' deletions of PSA using a constant 5' primer and five individual 3' primers starting at 736 bp, 610 bp, 505 bp, and 394 bp. The recombinant PSA proteins encoded 261, 231, 189, 154 and 117 amino acids. PSA-specific Class I- and II-restricted T cell hybridomas were generated by fusing Thy-1+tumor infiltrating lymphocytes (TIL) isolated from BALB/c mice challenged with Line 1/PSA/IL-2 tumors to the T cell fusion partner BWZ.36. MHC Class I (PSA 188-197) and Class II (PSA 238-253) T cell epitopes were identified.
[0168]The amino acid sequence of prostate specific antigen (PSA) as obtained from GenBank 45021731 kallikrein 3 is presented in Table 11.1. A cDNA vaccine for this antigen is available (Kim J J. Oncogene. 1998 17:3125-35). Predicted MHC Class II-presented epitopes of PSA are listed in Table 11.2. Experimentally defined MHC Class II-presented epitopes of PSA are listed in Table 11.3. Ii-Key/PSA hybrids containing some of the MHC Class II-presented epitopes of Tables 11.2 and 11.3 are listed in Table 11.4. Predicted MHC Class I-presented epitopes of PSA are listed in Table 11.5. Experimentally defined MHC Class I-presented epitopes of PSA are listed in Table 11.6. Ii-Key/PSA MHC II-presented epitope/PSA MHC I-presented epitope hybrids are listed in Table 11.7.
TABLE-US-00056 TABLE 11.1 Deduced amino acid sequence of PSA. 1 mwvpvvfltl svtwigaapl ilsrivggwe cekhsqpwqv lvasrgravc (SEQ ID NO: 358) 51 ggvlvhpqwv ltaahcirnk svillgrhsl fhpedtgqvf qvshsfphpl 101 ydmsllknrf lrpgddsshd lmllrlsepa eltdavkvmd lptqepalgt 151 tcyasgwgsi epeefltpkk lqcvdlhvis ndvcaqvhpq kvtkfmlcag 201 rwtggkstcs gdsggplvcn gvlqgitswg sepcalperp slytkvvhyr 251 kwikdtivan p
TABLE-US-00057 TABLE 11.2 Predicted MHC Class II-presented epitopes of PSA. PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO: 11.2.1 59 WVLTAAHCI 8.80 -- 359 11.2.2 2 WVPVVFLTL 8.10 -- 360 11.2.3 124 LRLSEPAEL 6.60 -- 361 11.2.4 67 IRNKSVILL 5.40 8 362 11.2.5 74 LLGRHSLFH 5.20 -- 363 11.2.6 72 VILLGRHSL 4.70 -- 364 11.2.7 223 LQGITSWGS 4.58 -- 365
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00058 TABLE 11.3 Experimentally defined MHC Class II-presented epitopes of PSA. SEQ ID PEPTIDE Pos. Sequence Ii-Key NO: 11.3.1 238 ERPSLYTKVVHYRKWI -- 366
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Peptide 11.3.1 was experimentally defined (Turner M J. J Immunol Meth. 2001 256:107-19).
TABLE-US-00059 TABLE 11.4 Ii-Key/PSA hybrids containing some of the MHC Class II-presented epitopes of Tables 11.2 and 11.3. PEPTIDE Pos. Sequence SEQ ID NO: A. Non-overlapping epitopes 11.4.1 2 Ac-LRMK-ava-WVPVVFLTL-NH2 367 11.4.2 124 Ac-LRMK-ava-LRLSEPAEL-NH2 368 11.4.3 223 Ac-LRMK-ava-LQGITSWGS-NH2 369 B. Overlapping epitopes 11.4.4 59 Ac-LRMK-ava- 370 WVLTAAHCI-NH2 11.4.5 67 Ac-LRMK-ava- 371 IRNKSVILL-NH2 11.4.6 72 Ac-LRMK-ava- 372 VILLGRHSL-NH2 11.4.7 74 Ac-LRMK-ava- 373 LLGRHSLFH-NH2 11.4.8 59, Ac-LRMK-ava- 374 67, WVLTAAHCIRNKSVILLGR 72, HSLFH-NH2 74
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 11.2 and 11.3. Peptide 11.4.8 contains several MHC Class II-presented epitopes each beginning at residue positions 59, 67, 72 and 74.
TABLE-US-00060 TABLE 11.5 Predicted MHC Class I-presented epitopes of PSA. PEPTIDE Pos. Sequence Score SEQ ID NO: 11.5.1 46 GRAVCGVL 2000 375 11.5.2 67 IRNKSVILL 2000 376 11.5.3 124 LRLSEPAEL 2000 377 11.5.4 18 APLILSRIV 660 378 11.5.5 7 FLTLSVTWI 607 379 11.5.6 249 YRKWIKDTI 600 380
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. The MHC Class I-presented epitopes were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptides 11.5.1, 11.5.2, 11.5.3 and 11.5.6 are presented optimally by HLA-B*2705. Peptide 11.5.4 is presented best by HLA-B*5102 and Peptide 11.5.5 is presented best by HLA-A*0201.
TABLE-US-00061 TABLE 11.6 Experimentally defined MHC Class I-presented epitopes of PSA. PEPTIDE Pos. Sequence SEQ ID NO: 11.6.1 188 HPQKVTKFML 381
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class I-presented epitope.
TABLE-US-00062 TABLE 11.7 Ii-Key/PSA MHC II-presented epitope/PSA MHC I- presented epitope hybrids. A Overlapping epitopes PEPTIDE Pos. Sequence SEQ ID NO: 11.7.1 2 Ac-LRMK-ava- 382 WVPVVFLTLSVTWI- NH2 11.7.2 67 Ac-LRMK-ava- 383 IRNKSVILL-NH2 11.7.3 124 Ac-LRMK-ava- 384 LRLSEPAEL-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 11.2 and a MHC Class I epitope of Table 11.5. In each of the example the sequences of the predicted MHC Class II and MHC Class I peptides overlap precisely. The residue position of the first amino acid in the predicted MHC Class II epitopes are reported.
Example 12
Ii-Key/Melanocyte Protein Pmel 17 Antigenic Epitope Hybrids
[0169]Melanoma is a leading target in the development of therapeutic peptide and DNA vaccines because several specific tumor-associated antigens have been identified, efficiency of vaccinating mice with peptide or DNA vaccines in treating melanoma is proved, and use of comparable vaccines in the clinic has had occasionally promising results. The use of Ii-Key/melanoma antigenic epitope hybrids in melanoma vaccination is considered in respective Examples concerning melanocyte protein Pme117, gp100, tyrosinase, and tyrosinase-s related protein.
[0170]Storkus and colleagues identified several MHC Class II-presented epitopes of gp 100/pme117 and tyrosinase melanocyte-associated antigens and tested the response of tumor-reactive human CD4+ T cells from various melanoma patients against these peptides (Kierstead L S. Br J. Cancer. 2001 85:1738-45). Two known and three novel CD4+ T cell epitopes were found using an IFN-gamma ELISPOT assay. Often freshly-isolated PBMC from HLA-DR4+ melanoma patients that are currently disease-free reveal elevated Th1-type CD4+ T-cells that recognize these peptides. Ii-Key/antigenic epitope hybrids incorporating these epitopes are presented in this Disclosure.
[0171]One problem in tumor immunotherapy is the fact that hosts can be tolerized to self proteins of the tumor. Intracutaneous immunization of C57BL/6 mice with a human Pme117/gp100 DNA vaccine, but not the murine DNA, induces T cell-mediated B16 melanoma protection in vivo (Wagner S N. J Invest Dermatol. 2000 115:1082-7). This state of unresponsiveness to the autoantigen Pme117/gp100 was broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. Mice receiving of Pme117/gp100 DNA mounted an antigen-specific cytotoxic T lymphocyte response to M3 melanoma. Furthermore M3 tumors growing in immunized mice lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pme117/gp100-positive. Ii-Key/antigenic epitope hybrids with appropriate immunization schemes and adjuvants can preferentially induce a Th1 or Th2 pattern of response thereby breaking tolerance.
[0172]The amino acid sequence of melanocyte protein Pmel 17 was obtained at NCBI, >gi|1125063|gb|AAB00386.11 melanocyte protein Pmel 17 [Homo sapiens]=>gi|639590|gb|AAC60634.1 gp100[Homo sapiens].
TABLE-US-00063 TABLE 12.1 Deduced amino acid sequence of gp 100/pmel. 1 mdlvlkrcll hlavigalla vgatkvprnq dwlgvsrqlr tkawnrqlyp (SEQ ID NO: 385) 51 ewteaqrldc wrggqvslkv sndgptliga nasfsialnf pgsqkvlpdg 101 viwvnntii ngsqvwggqp vypqetddac ifpdggpcps gswsqkrsfv 151 yvwktwgqyw qvlggpvsgl sigtgramlg thtmevtvyh rrgsrsyvpl 201 ahsssaftit dqvpfsvsvs qlraldggnk hflrnqpltf alqlhdpsgy 251 laeadlsytw dfgdssgtli sralvvthty lepgpvtaqv vlqaaiplts 301 cgsspvpgtt dghrptaeap nttagqvptt evvgttpgqa ptaepsgtts 351 vqvpttevis tapvqmptae stgmtpekvp vsevmgttla emstpeatgm 401 tpaevsivvl sgttaaqvtt tewvettare lpipepegpd assimstesi 451 tgslgplldg tatlrlvkrq vpldcvlyry gsfsvtldiv qgiesaeilq 501 avpsgegdaf eltvscqggl pkeacmeiss pgcqppaqrl cqpvlpspac 551 qlvlhqilkg gsgtyclnvs ladtnslavv stqlimpgqe aglgqvpliv 601 gillvlmavv lasliyrrrl mkqdfsvpql phssshwlrl prifcscpig 651 enspllsgqq v
TABLE-US-00064 TABLE 12.2 Predicted MHC Class II-presented epitopes of gp 100. PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO: 12.2.1 150 VYVWKTWGQ 6.80 -- 386 12.2.2 423 WVETTREL 6.40 -- 387 12.2.3 477 LYRYGSFSV 6.40 3 388 12.2.4 290 VVLQAAIPL 6.40 10 389 12.2.5 552 LVLHQILKG 6.10 -- 390 12.2.6 596 VPLIVGILL 5.80 -- 391 12.2.7 600 VGILLVLMA 5.80 -- 392 12.2.8 605 VLMAVVLAS 5.80 -- 393 12.2.9 604 LVLMAVVLA 5.70 -- 394 12.2.10 3 LVLKRCLLH 5.40- -- 395 7.20 12.2.11 615 IYRRRLMKQ 5.10- -- 396 5.70 12.2.12 616 YRRRLMKQD 4.50 -- 397 12.2.13 48 LYPEWTEAQ 4.30 8 398
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00065 TABLE 12.3 Experimentally defined MHC Class II-presented epitopes of gp100. SEQ ID PEPTIDE Pos. Sequence Ii-Key NO: 12.3.1 44 WNRQLYPEWTEAQRLD 4 399 12.3.2 615 IYRRRLMKQDFSVPQLPHS -- 400 12.3.3 576 SLAVVSTQLIMPG -- 401 12.3.4 175 GRAMLGTHTMEVTVY -- 402 12.3.5 74 GPTLIGANASFSIALN -- 403
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. DR*0401 best presented peptide 12.3.1 (Storkus W. Forum (Genova). 2000 10:256-70) and peptide 12.3.2 (Kierstead L. Brit J Cancer. 2001 85:1738-45). The remaining peptides of this Table were identified by Kobayashi H. (Cancer Res. 2001 61:7577-84).
TABLE-US-00066 TABLE 12.4 Ii-Key/gp 100 hybrids containing some of the MHC Class II-presented epitopes of Table 12.2 and 12.3. SEQ ID PEPTIDE Pos. Sequence NO: A. Non-overlapping epitopes 12.4.1 615 Ac-LRMK-ava- 404 IYRRRLMKQDFSVPQLPHS-NH2 12.4.2 3 Ac-LRMK-ava-LVLKRCLLH-NH2 405 12.4.3 150 Ac-LRMK-ava-VYVWKTWGQ-NH2 406 12.4.5 423 Ac-LRMK-ava-WVETTAREL-NH2 407 12.4.6 477 Ac-LRMK-ava-LYRYGSFSV-NH2 408 B. Overlapping epitopes 12.4.7 44 Ac-LRMK-ava- 409 WNRQLYPEWTEAQRLD-NH2 12.4.8 48 Ac-LRMK-ava-LYPEWTEAQ-NH2 410 12.4.9 44, 48 Ac-LRMK-ava- 411 WNRQLYPEWTEAQRLD-NH2 12.4.10 596 Ac-LRMK-ava-VPLIVGILL-NH2 412 12.4.11 600 Ac-LRMK-ava-VGILLVLMA-NH2 413 12.4.12 605 Ac-LRMK-ava-VLMAVVLAS-NH2 414 12.4.13 596, 600, 605 Ac-LRMK-ava- 415 VPLIVGILLVLMAVVLAS-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2.
TABLE-US-00067 TABLE 12.5 Predicted MHC Class I-presented epitopes of gp 100. PEPTIDE Pos. Sequence Score SEQ ID NO: 12.5.1 619 RLMKQDFSV 1495 416 12.5.2 520 LPKEACMEI 629 417 12.5.3 602 ILLVLMAVV 412 418 12.5.4 479 RYGSFSVTL 400 419 12.5.5 154 KTWGQYWQV 315 420 12.5.6 17 ALLAVGATK 45 421 12.5.7 614 LIYRRRLMK 20 422
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. The MHC Class I-presented epitopes were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptides 12.5.1, 12.5.3 and 12.5.5 are presented by HLA-A*0201. Peptide 12.5.2 is presented by HLA-B*5101. Peptides 12.5.4 is presented by HLA-A*24. Peptides 12.5.6 and 12.5.7 are presented by HLA-A3.
TABLE-US-00068 TABLE 12.6 Experimentally defined MHC Class I-presented epitopes of gp100. PEPTIDE Pos. Sequence SEQ ID NO: 12.6.1 280 YLEPGPVTA 423 12.6.2 17 ALLAVGATK 424 12.6.3 209 ITDQVPFSV 425 12.6.4 614 LIYRRRLMK 426 12.6.5 619 RLMKQDFSV 427 12.6.6 639 RLPRIFCSC 428 12.6.7 154 KTWGQYWQV 429 12.6.8 177 AMLGTHTMEV 430 12.6.9 570 SLADTNSLAV 431 12.6.10 70 VSNDGPTLI 432 12.6.11 87 ALNFPGSQK 433
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. Peptide 12.6.1 is presented by HLA-A2 (Slingluff C. Clin Cancer Res. 2001 7:3012-24). Peptide 12.6.2 is presented by HLA-A3 (Yamshchikov G. Int J Cancer. 2001 92:703-11). Peptides 12.6.3, 12.6.5, 12.6.6 and 12.6.7 are presented by HLA-A*02012 (Kawakami Y. Proc Natl Acad Sci USA 1998). Peptide 12.6.8 and 12.6.9 are presented by HLA-A*0201 (Tsai V. J Immunol. 1997 158:1796-802). Peptide 12.6.10 is presented by HLA-Cw8 (Castelli C. J Immunol. 1999 162:1739-48). Peptide 12.6.11 is presented by HLA-A3 and HLA-A11.
TABLE-US-00069 TABLE 12.7 Designed Ii-Key/gp 100 hybrids containing some of the MHC Class I- and Class II-presented epitopes of Tables 4 and 5. SEQ ID PEPTIDE Pos. Sequence NO: A. Non-overlapping epitopes 12.7.1 II: 520, Ac-LRMK-ava-LPKEACMEI- 434 I: 552 LVLHQILKG-NH2 12.7.2 II: 17, Ac-LRMK-ava-ALLAVGATK- 435 I: 3 LVLKRCLLH-NH2 B. Overlapping epitopes 12.7.3 II: 570, Ac-LRMK-ava- 436 I: 576 SLADTNSLAVVSTQLIMPG-NH2 12.7.4 II: 177, Ac-LRMK-ava- 437 I: 175 GRAMLGTHTMEVTVY-NH2 12.7.5 II: 70, Ac-LRMK-ava- 438 II87, VSNDGPTLIGANASFSIALNFPGSQK- I: 74 NH2 12.7.6 II: 614 Ac-LRMK-ava- 439 (619), LIYRRRLMKQDFSVPQLPHS-NH2 I: 615 12.7.7 II: 154, Ac-LRMK-ava-VYVKTWGQYWQV-NH2 440 I: 150 12.7.8 II: 479, Ac-LRMK-ava-LYRYGSFSVTL-NH2 441 I: 477
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope, with MHC Class II indicated as I:, and MHC Class II indicated as II:. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2. Peptides 12.7.3, 12.7.4 and 12.7.5 have been already proposed (Kabayashi H. Cancer Res. 2001 61:7577-84). Peptide 12.7.6--amino acid sequence of both MHC Class I- and II-presented gp 100 epitopes are experimentally defined and coincide.
Example 13
Ii-Key/Tyrosinase-Related Protein 2 Antigenic Epitope Hybrids
[0173]The amino acid sequence of tyrosinase-related protein 2 as given in GenBank gi|731026|sp|P40126|TYR2_HUMAN Dopachrome tautomerase precursor (DT) (DCT) (Dopachrome delta-isomerase) (Tyrosinase-related protein 2) (TRP-2) (TRP2) is presented in Table 13.1. Predicted MHC Class II-presented epitopes of TRP-2 are listed in Table 13.2. Designed Ii-Key/TRP-2 antigenic epitope hybrids containing some of the MHC Class II-presented epitopes of Table 13.2 are listed in Table 13.3. Predicted MHC Class-I presented epitopes of TRP-2 are listed in Table 13.4. Experimentally defined MHC Class I-presented TRP-2 epitopes are listed in Table 13.5. Designed Ii-Key/TRP-2 hybrids containing some of the MHC Class I- and II-presented epitopes of Tables 13.2, 13.3, 13.4 and 13.5 are listed in Table 13.6.
TABLE-US-00070 TABLE 13.1 Deduced amino acid sequence of melanocyte protein Pmel 17. (SEQ ID NO: 442) 1 msplwwgfll sclgckilpg aqgqfprvcm tvdslvnkec cprlgaesan 51 vcgsqqgrgq ctevradtrp wsgpyilrnq ddrelwprkf fhrtckctgn 101 fagyncgdck fgwtgpncer kkppvirqni hslspqereq flgaldlakk 151 rvhpdyvitt qhwlgllgpn gtqpqfancs vydffvwlhy ysvrdtllgp 201 grpyraidfs hqgpafvtwh ryhllclerd lqrlignesf alpywnfatg 251 rnecdvctdq lfgaarpddp tlisrnsrfs swetvcdsld dynhlvtlcn 301 gtyegllrrn qmgrnsmklp tlkdirdcls lqkfdnppff qnstfsfrna 351 legfdkadgt ldsqvmslhn lvhsflngtn alphsaandp ifvvlhsftd 401 aifdewmkrf nppadawpqe lapighnrmy nmvpffppvt neelfltsdq 451 lgysyaidlp vsveetpgwp ttllvvmgtl valvglfvll aflqyrrlrk 501 gytplmethl sskryteea
TABLE-US-00071 TABLE 13.2 Predicted MHC Class II-presented epitopes of TRP-2. PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO: 13.2.1 156 YVITTQHWL 8.20 9 443 13.2.2 451 LGYSYAIDL 7.60 -- 444 13.2.3 64 VRADTRPWSG 6.60 -- 445 13.2.4 483 LVGLFVLLA 6.10 -- 446 13.2.5 272 LISRNSRFS 5.70 -- 447 13.2.6 392 FVVLHSFTD 5.50 -- 448 13.2.7 219 WHRYHLLCL 5.40 7 449 13.2.8 498 LRKGYTPLM 5.30 -- 450 13.2.9 365 VMSLHNLVH 5.20 -- 451 13.2.10 474 LVVMGTLVA 5.10 -- 452
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00072 TABLE 13.3 Designed Ii-Key/TRP-2 antigenic epitope hybrids containing some of the MHC Class II-presented epitopes of Table 13.2. PEPTIDE Pos. Sequence Score SEQ ID NO: 13.3.11 156 Ac-LRMK-ava- 453 YVITTQHWL-NH2 13.3.12 451 Ac-LRMK-ava- 454 LGYSYAIDL-NH2 13.3.13 64 Ac-LRMK-ava- 455 VRADTRPSG-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 13.2.
TABLE-US-00073 TABLE 13.4 Predicted MHC Class-I presented epitopes of TRP 2. PEPTIDE Pos. Sequence Score SEQ ID NO: 13.4.1 277 SRFSSWETV 3000 456 13.4.2 408 KRFNPPADA 3000 457 13.4.3 325 IRDCLSLQK 2000 458 13.4.4 150 KRVHPDYVI 1800 459 13.4.5 427 NRMYNMVPF 1000 460 13.4.6 485 GLFVLLAFL 999 461 13.4.7 180 SVYDFFVWL 973 462 13.4.8 490 LAFLQYRRL 665 463 13.4.9 431 NMVPFFPPV 363 464 13.4.10 185 FVWLHYYSV 348 465 13.4.11 180 SVYDFFVWL 504 466 13.4.12 199 GPGRPYRAI 440 467 13.4.13 264 AARPDDPTL 360 468 13.4.14 353 GFDKADGTL 330 469 13.4.15 408 KRFNPPADA 300 470 13.4.16 189 HYYSVRDTL 280 471 13.4.17 331 LQKFDNPPF 240 472
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. The MHC Class I-presented epitopes of Table 9.4 were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptides 13.4.1, 13.4.2, 13.4.3, 13.4.4 and 13.4.5 are presented by HLA-B*2705. Peptides 13.4.6, 13.4.7, 13.4.9 and 13.4.10 are presented by HLA-A*0201. Peptides 13.4.8 is presented by HLA-B*5102. Peptide 13.4.11 is presented by HLA-A*0205. Peptides 13.4.12 is presented by HLA-B5101 and HLA-B*5102. Peptide 13.4.13 is presented by HLA-B7. Peptides 13.4.14 is presented by Cw*0401. Peptide 13.4.15 is presented by HLA-B*2702. Peptide 13.4.16 is presented by HLA-A24. Peptide 13.4.17 is presented by HLA-B62.
TABLE-US-00074 TABLE 13.5 Experimentally defined MHC Class I-presented TRP-2 epitopes. PEPTIDE Pos. Sequence SEQ ID NO: 13.5.1 180 SVYDFFVWL 473 13.5.2 360 TLDSQVMSL 474 13.5.3 288 SLDDYNHLV 475 13.5.4 455 YAIDLPVSV 476
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class I-presented epitope. Peptide 13.5.1 is presented by HLA-A2 (Parkhurst M R. Cancer Res. 1998 58:4895-901). Peptides 13.5.2 and 13.5.3 are presented by HLA-A2.1 (Noppen C. Int J Cancer. 2000 87:241-6). Peptide 13.5.4 is presented by HLA-A2.1 (Harada M. Cancer Res. 2001 61:1089-94).
TABLE-US-00075 TABLE 13.6 Designed Ii-Key/TRP-2 hybrids containing some of the MHC Class I and II-presented epitopes of Tables 13.2, 13.3, 13.4 and 13.5. SEQ ID PEPTIDE Pos. Sequence NO: 13.6.1 I: 180; Ac-LRMK-ava-YVITTQHWL- 477 II: 156 SVYDFFVWL-NH2 13.6.2 I: 455; Ac-LRMK-ava-LGYSYAIDLPVSV-NH2 478 II: 451 13.6.3 I: 360; Ac-LRMK-ava-TLDSQVMSLHNLVH-NH2 479 II: 365
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 13.2 and a MHC Class I epitope of Table 13.4.
Example 14
Ii-Key/Melanoma Tyrosinase Antigenic Epitope Hybrids
[0174]Tyrosinase has many advantages as a target antigen for the immunotherapy of patients with melanoma because it is expressed in nearly all melanoma specimens with a high degree of cellular homogeneity, and its distribution in normal tissues is limited to melanocytes. Several MHC Class I-presented epitopes have been identified and used clinically, and MHC Class II-presented epitopes have been discovered. The following summaries of the current state-of-the-art in identification and use of peptide vaccines, DNA vaccines, and dendritic cell charging with peptide preparations (tumor cell lysates) are presented in part to illustrate the value of the products and methods of this Disclosure to improving these procedures.
[0175]Rosenberg and colleagues identified a HLA-A2.1-presented restricted melanoma tyrosinase epitope (tyrosinase8-17; CLLWSFQTSA) (SEQ ID NO: 480) (Riley J P. J Immunother. 2001 24:212-20). In this study, the comparative binding to HLA-A2.1 of a series of algorithm-predicted peptides versus that of a standard peptide with an intermediate binding affinity was determined. Twelve peptides with binding affinities within 80% of that of the standard peptide stimulated peripheral blood mononuclear cells (PBMC) in vitro from three HLA-A2.1+ patients with metastatic melanoma. PBMC from 23 HLA-A2.1+ patients were stimulated in vitro with tyrosinase:8-17. Eleven bulk T-cell cultures demonstrated specific peptide recognition, and six of these also recognized HLA-A2.1+ tyrosinase+ melanoma cells. This epitope can be incorporated in an Ii-Key/MHC Class II-presented epitope/MHC Class I-presented epitope hybrid.
[0176]Weber and colleagues found that patients with resected melanoma mounted an immune response against gp100(209-217)(210M) (IMDQVPSFV) (SEQ ID NO: 481) and tyrosinase(368-376)(370D) (YMDGTMSQV) (SEQ ID NO: 482), emulsified with incomplete Freund's adjuvant (Lee P. J Clin Oncol. 2001 19:3836-47). Patients received peptides/IFA with or without IL-12 (30 ng/kg) to evaluate the toxicities and immune responses. Immunizations were administered every 2 weeks for 8 weeks, then every 4 weeks for 12 weeks, and then once 8 weeks later. Thirty-four of 40 patients developed a positive skin test response to the gp100 peptide but none responded to the tyrosinase peptide. Immune responses were measured by release of gamma-interferon in an enzyme-linked immunosorbent assay (ELISA) by effector cells in the presence of peptide-pulsed antigen-presenting cells or by an antigen-specific tetramer flow cytometry assay. Thirty-three of 38 patients demonstrated an immune response by ELISA after vaccination, as did 37 of 42 patients by tetramer assay. Twenty-four of 48 patients relapsed with a median follow-up of 20 months, and 10 patients in this high-risk group have died.
[0177]Slingluff and colleagues evaluated peptide vaccine immunogenicity of several peptides restricted to different HLA-A alleles in draining lymph nodes and peripheral blood of melanoma patients because vaccine trials have been limited mostly to those associated with HLA-A2, and immune responses have been detected inconsistently (Yamshchikov G V. Int J Cancer. 2001 92:703-11). They vaccinated stage 1V melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA-A1 (DAEKSDICTDEY) (SEQ ID NO: 483), HLA-A2 (YLEPGPVTA (SEQ ID NO: 484) and YMDGTMSQV (SEQ ID NO: 485)) and HLA-A3 (ALLAVGATK) (SEQ ID NO: 486) in an emulsion with GM-CSF and Montanide ISA-51 adjuvant. CTL responses to vaccinating peptides were found in a lymph node draining a vaccine site (sentinel immunized node, SIN) in 5/5 patients (100%) in PBLs of 2/5 patients (40%). Peptides restricted by HLA-A1 and -A3 and HLA-A2 restricted peptide, YMDGTMSQV (SEQ ID NO: 485), were immunogenic.
[0178]Cytotoxic T lymphocytes against melanoma-associated antigens were induced by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo (Oertli D. Hum Gene Ther. 2002 13:569-75). Patients received psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Subsequent boosts used corresponding synthetic nona-peptides and GM-CSF. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detected in three of three patients. During the vaccination treatment, antigen-specific CTL frequencies exceeding 1:10,000 peripheral CD8+ T cells could be observed.
[0179]Two stage 1V melanoma patients vaccinated with an HLA-A2- or HLA-A24-restricted tyrosinase peptide, and GM-CSF had long-term freedom from recurrence (Scheibenbogen C. Int J Cancer. 2002 99:403-8). While the patients had experienced 9 and 12 relapses (mostly subcutaneous), respectively, during the 3 years before vaccination, they experienced freedom from relapse for more than 2 years after vaccination. T-cell responses to the vaccine peptide were found in the peripheral blood of both patients using an IFN-gamma ELISPOT assay.
[0180]Mule and colleagues found that addition of keyhole limpet hemocyanin (KLH) augmented the efficacy of both tumor lysate-pulsed dendritic cells and peptide-pulsed dendritic cells immunizations for immune priming and rejection of established metastases of the D5 subline of B16 melanoma in vivo (Shimizu K. Cancer Res. 2001 61:2618-24). Interleukin 2 further augmented the enhancement afforded by KLH, as measured by cure rates and overall survival, in the absence of autoimmune depigmentation. KLH added to dendritic cells immunizations markedly enhances tumor-specific T cell production of IFN-gamma. D5 melanoma exposed to similar levels of IFN-gamma results in substantial expression of MHC Class I molecules. Immunization with dendritic cells pulsed with KLH and mouse tyrosinase-related protein-2 peptide results in enhanced reduction of B16 melanoma metastases; the effect is most pronounced in a setting where tyrosinase-related protein-2 peptide-pulsed dendritic cells alone are completely ineffective.
[0181]Therapeutic efficacy of a tumor cell-based vaccine against B16 melanoma requires disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte-associated antigen (CTLA)-4 pathway or the CD25+ regulatory T cells. Combination of CTLA-4 blockade and depletion of CD25+ T cells results in maximal tumor rejection (Sutmuller R P. J Exp Med. 2001 194:823-32). Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2(180-188)-specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8+ T cell subset. The CTL response against melanoma antigens is an important component of the therapeutic antitumor response, and the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25+ T cells indicates that CD25+ T cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is, therefore, a promising concept for the induction of therapeutic antitumor immunity.
[0182]The amino acid sequence of tyrosinase as given in GenBank 4507753|ref|NP--000363.1| tyrosinase (oculocutaneous albinism IA); Tyrosinase [Homo sapiens] is listed in Table 14.1. Predicted MHC Class II-presented epitopes of tyrosinase are listed in Table 14.2. Experimentally defined MHC Class II-presented epitopes of tyrosinase are listed in Table 14.3. Designed Ii-Key/tyrosinase hybrids containing some of the MHC Class II-presented epitopes of Table 14.2 and 14.3 are listed in Table 14.4. Predicted MHC Class I-presented epitopes of tyrosinase are listed in Table 14.5. The experimental identification of MHC Class I-presented epitopes of tyrosinase (Pos. 240, 368, 146) is described in the gp100 example. Experimentally defined MHC Class I-presented epitopes of tyrosinase are listed in Table 14.6. Designed Ii-Key/tyrosinase hybrids containing some of the MHC Class I- and MHC Class II-presented epitopes of Tables 14.2, 14.3, 14.4 and 14.5 are listed in Table 14.7.
TABLE-US-00076 TABLE 14.1 Deduced amino acid sequence of tyrosinase. 1 mllavlycll wsfqtsaghf pracvssknl mekeccppws gdrspcgqls (SEQ ID NO: 487) 51 grgscqnill snaplgpqfp ftgvddresw psvfynrtcq csgnfmgfnc 101 gnckfgfwgp ncterrllvr rnifdlsape kdkffayltl akhtissdyv 151 ipigtygqmk ngstpmfndi niydlfvwmh yyvsmdallg gseiwrdidf 201 aheapaflpw hrlfllrweq eiqkltgden ftipywdwrd aekcdictde 251 ymggqhptnp nhlspasffs swqivcsrle eynshqslcn gtpegplrrn 301 pgnhdksrtp rlpssadvef clsltqyesg smdkaanfsf rntlegfasp 351 ltgiadasqs smhnalhiym ngtmsqvqgs andpifllhh afvdsifeqw 401 lrrhrplqev ypeanapigh nresymvpfi plyrngdffi sskdlgydys 451 ylqdsdpdsf qdyiksyleq asriwswllg aamvgavlta llaglvsllc 501 rhkrkqlpee kqpllmeked yhslyqshl
TABLE-US-00077 TABLE 14.2 Predicted MHC Class II-presented epitopes of tyrosinase. PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO: 14.2.1 401 LRRHRPLQE 6.60 6 488 14.2.2 179 MHYYVSMDA 6.40 -- 489 14.2.3 400 WLRRHRPLQ 6.25 5 490 14.2.4 118 LVRRNIFDL 5.70 9 491 14.2.5 366 LHIYMNGTM 5.50 -- 492 14.2.6 368 IYMNGTMSQ 5.40 -- 493 14.2.7 182 YVSMDALLG 5.50 -- 494 14.2.8 150 VIPIGTYGQ 5.50 7 495 14.2.9 338 FSFRNTLEG 5.40 -- 496 14.2.10 498 LLCRHKRKQ 5.30 -- 497 14.2.11 1 MLLAVLYCL 5.20 -- 498 14.2.12 167 FNDINIYDL 5.20 -- 499
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00078 TABLE 14.3 Experimentally defined MHC Class II-presented epitopes of tyrosinase. SEQ ID PEPTIDE Pos. Sequence Ii-Key NO: 14.3.1 56 QNILLSNAPLGPQFP -- 500 14.3.2 365 ALHIYMNGTMSQVQGSA -- 501 14.3.3 156 YGQMKNGSTPMFNDINIYDL -- 502
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Peptide 14.3.1 is presented by HLA-DR*0401 (Storkus W. Forum (Genova). 2000 10:256-270). Peptides 14.3.2 and 14.3.3 are presented by HLA-DR*0401 (Kierstead L. Brit J Cancer. 2001 85:1738-45). Peptide 14.3.2 contains an N-glycosylation site.
TABLE-US-00079 TABLE 14.4 Designed Ii-Key/tyrosinase hybrids containing some of the MHC Class II-presented epitopes of Table 14.2 and 14.3. SEQ ID PEPTIDE Pos. Sequence NO: Non-overlapping 14.4.1 56 Ac-LRMK-ava- 503 QNILLSNAPLGPQFP-NH2 14.4.2 118 Ac-LRMK-ava- 504 LVRRNIFDL-NH2 14.4.3 338 Ac-LRMK-ava- 505 FSFRNTLEG-NH2 14.4.4 498 Ac-LRMK-ava- 506 LLCRHKRKQ-NH2 Overlapping epitopes 14.4.5 365 Ac-LRMK-ava- 507 ALHIYMNGTMSQVQGSA-NH2 14.4.6 366 Ac-LRMK-ava-LHIYMNGTM-NH2 508 14.4.7 368 Ac-LRMK-ava-IYMNGTMSQ-NH2 509 14.4.8 365 366 and Ac-LRMK-ava-ALHIYMNGTMSQ- 510 368 NH2 14.4.9 182 Ac-LRMK-ava-YVSMDALLG-NH2 511 14.4.10 179 Ac-LRMK-ava-MHYYVSMDA-NH2 512 14.4.11 179 and 182 Ac-LRMK-ava-MHYYVSMDALLG- 513 NH2 14.4.12 150 Ac-LRMK-ava-VIPIGTYGQ-NH2 514 14.4.13 156 Ac-LRMK-ava- 515 YGQMKNGSTPMFNDINIYDL-NH2 14.4.14 167 Ac-LRMK-ava-FNDINIYDL-NH2 516 14.4.15 150 156 and Ac-LRMK-ava- 517 167 VIPIGTYGQMKNGSTPMFNDINIYDL- NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2.
TABLE-US-00080 TABLE 14.5 Predicted MHC Class I-presented epitopes of tyrosinase. PEPTIDE Pos. Sequence Score SEQ ID NO: 14.5.1 243 KCDICTDEY 25.0 518 14.5.2 369 YMNGTMSQV 531.4 519 14.5.3 1 MLLAVLYCL 309.1 520 14.5.4 207 FLPWHRLFL 540.5 521
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. The MHC Class I-presented epitopes of Table 9.4 were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptide 14.4.1 is presented by HLA-A1. Peptides 14.5.2, 14.5.3 and 14.5.4 are presented by HLA*A0201.
TABLE-US-00081 TABLE 14.6 Experimentally defined MHC Class I-presented epitopes of tyrosinase. PEPTIDE Pos. Sequence SEQ ID NO: 14.6.1 240 DAEKSDICTDEY 522 14.6.2 368 YMDGTMSQV 523 14.6.3 146 SSDYVIPIGTY 524
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Peptide 14.6.1 is presented by HLA-A1 (Yamshchikov G. Int J Cancer. 2001 92:703-11). Peptide 14.6.2 is presented by HLA-A2 (Yamshchikov G. Int J Cancer. 2001 92:703-11). Peptide 14.6.3 is presented by HLA-A1 (Kawakami Y. J Immunol. 1998 161:6985-92).
TABLE-US-00082 TABLE 14.7 Designed Ii-Key/tyrosinase hybrids containing some of the MHC Class I- and MHC Class II- presented epitopes of Tables 14.2, 14.3, 14.5, and 14.6). SEQ ID PEPTIDE Pos. Sequence NO: Non-overlapping epitopes 14.7.1 240 Ac-LRMK-ava- 525 and DAEKSDICTDEY- 56 QNILLSNAPLGPQFP-NH2 14.7.2 207 Ac-LRMK-ava-FLPWHRLFL- 526 and LRRHRPLQE-NH2 401 Overlapping epitopes 14.7.3 368 (371D) Ac-LRMK-ava- 527 and 365 ALHIYMNGTMSQVQGSA-NH2 (366, 368) 14.7.4 146 and 156 Ac-LRMK-ava- 528 SSDYVIPIGTYGQMKNGSTPM FNDINIYDL-NH2 14.7.5 1 and 1 Ac-LRMK-ava- 529 MLLAVLYCL-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 14.2. PEPTIDE 14.7.5 includes amino acid sequences of both MHC Class I- and II-presented epitope of tyrosinase, which are experimentally defined and coincide.
Example 15
Ii-Key/melanoma Antigen MART-1 Antigenic Epitope Hybrids
[0183]Rosenberg and colleagues immunized metastatic melanoma patients with autologous dendritic cells presenting epitopes derived from the melanoma-associated antigens MART-1 and gp100 (Panelli M C. J Immunother. 2000 23:487-98). The DCs were generated by 5- to 7-day incubation in interleukin-4 (1,000 U/mL) and granulocyte-macrophage colony-stimulating factor (1,000 U/mL) of peripheral blood monocytes obtained by leukapheresis. Before administration, the DCs were pulsed separately with the HLA-A*0201-associated melanoma epitopes MART-1(27-35) and gp-100-209-2M. The DCs were administered four times at 3-week intervals. A first cohort of patients (n=3) was treated with 6×107 DCs and a second cohort (n=5) with 2×108 DCs (in either case, one half of the DCs were pulsed with MART-1(27-35) and the other half was pulsed with gp-100-209-2M). In a final cohort under accrual (n=2) 2×108 DCs were administered in combination with interleukin-2 (720,000 IU/kg every 8 hours). The recovery of DCs after in vitro culture ranged from 3% to 35% (mean, 15%) of the original peripheral blood monocytes. Administration of DCs caused no symptoms at any of the doses, and the concomitant administration of interleukin-2 did not cause toxicity other than that expected for interleukin-2 alone. Monitoring of patients' cytotoxic T lymphocyte reactivity before and after treatment revealed enhancement of cytotoxic T lymphocyte reactivity only in one of five patients tested. Of seven patients evaluated for response, one had a transient partial response with regression of pulmonary and cutaneous metastases.
[0184]Ioannides and colleagues demonstrated reduced recognition of metastatic melanoma cells by autologous MART-1 specific CTL correlated to TAP deficiency (Murray J L. J Immunother. 2000 23:28-35). Class I expression in context with T-cell receptor expression is crucial for peptide presentation and induction of CD8+ cytotoxic T lymphocytes (CTL). Presentation of MHC class I bound peptides depends on transporter-associated proteins (TAP) expression and function. Tumor infiltrating lymphocytes from a patient with melanoma were isolated, expanded in vitro in the presence of interleukin-2, and tested for cytotoxicity against HLA-A2 positive, MART-1 positive autologous tumor cells, an HLA-A2-positive, MART-1 positive melanoma cell line (MeI-501), and HLA-A2-negative melanoma cells. Significant killing occurred against both A2-positive cell lines (63% and 65%, respectively), but not against the A2-negative line (18%) or A2-positive autologous tumor (1.5%). These CTL preferentially recognized the MART-1 peptide F119, 27-35, and gp100 peptide F125, 280-288, resulting in a 30% to 60% enhancement of lysis when autologous tumor or major histocompatibility complex class I "empty" T2 cells were pulsed with either peptide. To address whether the deficiency in autologous tumor recognition might be related to a deficiency in Ag presentation, screening for the presence of TAP1 and TAP2 transcripts by polymerase chain reaction, Southern blotting, and scanning densitometry using sequence-specific primers and probes. Both TAP1 and TAP2 expression levels in the autologous tumor were minimal, yet were upregulated 7- to 18-fold, respectively, by interferon-gamma. Despite this increase, a similar increase in cytotoxicity did not occur. In short, deficiencies in TAP presentation may have functional significance for tumor escape from immunosurveillance and with respect to impending vaccine trials.
[0185]Slingluff and colleagues demonstrated terminal modifications inhibit proteolytic degradation of an immunogenic MART-1(27-35) peptide (Brinckerhoff L H. Int J Cancer 1999 Oct. 29; 83(3):326-34). The stability of the immunogenic peptide MART-1(27-35) in fresh normal human plasma (NHP) was tested to identify modifications protecting against enzymatic destruction without loss of immunogenicity. MART-1(27-35) peptide (AAGIGILTV) (SEQ ID NO: 530) and modified forms were incubated in plasma for varied time intervals and evaluated for their ability to reconstitute the epitope for MART-1(27-35)-reactive CTL. Loss of CTL reactivity signaled loss of immunoreactive peptide. When 1 microM MART-1(27-35) peptide was incubated in plasma prior to pulsing on target cells, CTL reactivity was lost within 3 hr, and the calculated half-life of this peptide was 22 sec. This degradation was mediated by peptidases. The stability of MART-1(27-35) was markedly prolonged by C-terminal amidation and/or N-terminal acetylation (peptide capping), or by polyethylene-glycol modification (PEGylation) of the C-terminus. These modified peptides were recognized by CTL.
[0186]Romero and colleagues demonstrated that CpG is an efficient adjuvant for specific CTL induction against tumor antigen-derived peptide (Miconnet I. J Immunol. 2002 168:1212-8). Mice transgenic for a chimeric MHC class I molecule were immunized with a peptide analog of MART-1/Melan-A(26-35) in the presence of CpG oligonucleotides alone or emulsified in IFA. The CTL response was monitored ex vivo by tetramer staining of lymphocytes. In blood, spleen, and lymph nodes, peptide mixed with CpG ODN alone was able to elicit a stronger systemic CTL response as compared with peptide emulsified in IFA. Moreover, CpG ODN in combination with IFA further enhanced the CTL response in terms of the frequency of tetramer+CD8+ T cells ex vivo. The CTL induced in vivo against peptide analog in the presence of CpG ODN are functional, as they were able to recognize and kill melanoma cells in vitro.
[0187]Mitchell and colleagues demonstrated synthetic insertion of signal sequences enhance MHC Class I presentation of a peptide from the melanoma antigen MART-1 (Minev B R. Eur J Immunol. 2000 30:2115-24). Addition of synthetic signal sequences at the N terminus, but not at the C terminus, of an epitope from the human melanoma antigen MART-1 enhanced its presentation in both TAP-deficient and TAP-expressing cells. A peptide construct, composed of the epitope replacing the hydrophobic part of a natural signal sequence, was also very effective. Interestingly, an artificial signal sequence containing the same epitope was the most efficient construct for enhancing its presentation. These peptide constructs facilitated epitope presentation when loaded into the cytosol of TAP-deficient T2 cells, TAP-expressing melanoma cells and human dendritic cells.
[0188]Zajac and colleagues demonstrated immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen (Schutz A. Cancer Gene Ther. 2001 8:655-61). The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding MART-1/Melan-A 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation. Enhanced further with Ii-RGC.
[0189]Falo and colleagues demonstrated direct transfection and activation of human cutaneous dendritic cells (Larregina A T. Gene Ther. 2001 8:608-17). A gene gun was used to transfect human skin organ cultures with a particular goal of expressing transgenic antigens in resident cutaneous dendritic cells. Gold particles delivered to human skin are observed primarily in the epidermis, even when high helium delivery pressures are used. Langerhans cells resident in the basal epidermis can be transfected, and gene gun delivery is sufficient to stimulate the activation and migration of skin dendritic cells. RT-PCR analysis of dendritic cells, which have migrated from transfected skin, demonstrates transgenic mRNA, indicating direct transfection of cutaneous dendritic cells. Transfected epidermal Langerhans cells can efficiently present a peptide derived from the transgenic melanoma antigen MART-1 to a MART-1-specific CTL.
[0190]Mule and colleagues demonstrated that administration of tumor lysate-pulsed DCs is nontoxic and capable of inducing immunological response to tumor antigen (Chang A E. Clin Cancer Res. 2002 8:1021-32). Fourteen patients with stage 1V solid malignancies were treated in cohorts that received 106, 107, and 108 dendtiric cells i.d. every 2 weeks for three vaccines. Each vaccine was composed of a mixture of half DCs pulsed with autologous tumor lysate and the other half with keyhole limpet hemocyanin (KLH). Local accumulation of CD4+ and CD8+ T cells were found at the vaccination sites. There was a significant proliferative response of PBMCs to KLH induced by the vaccine. In 5 of 6 patients, the vaccine resulted in increased IFN-gamma production by PBMCs to KLH in an ELISPOT assay. Using the same assay, 3 of 7 patients' PBMCs displayed increased IFN-gamma production in response to autologous tumor lysate. One patient with melanoma also was observed to have an increased frequency of MART-1- and gp100-reactive CD8(+) T cells after vaccination. By delayed-type hypersensitivity testing, 8 of 9 and 4 of 10 patients demonstrated reactivity to KLH and autologous tumor, respectively. Ii-Key/antigenic epitope hybrids will improve the efficiency of this immunopriming technology.
[0191]Kourilsky and colleagues demonstrated cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells (Motta I. J Immunol. 2001 167:1795-802). Recombinant canarypox virus (ALVAC) encoding the melanoma-associated Ag, Melan-A/MART-1 (MART-1), was tested in cancer immunotherapy, using a dendritic cell (DC)-based approach. ALVAC MART-1-infected DC express, and process and present, the antigen encoded by the viral vector. One consistent feature of infection by ALVAC was induction of apoptosis, and cross-presentation of Ag when uninfected DC are cocultured with ALVAC MART-1-infected DC. Uptake of apoptotic virally infected DC by uninfected DC and subsequent expression of tumor antigen in the latter were verified by flow cytometry analysis, image cytometry, and confocal microscopy. Functional activity was monitored in vitro by the stimulation of a MART-1-specific cytotoxic T cell clone. Heightened efficiency in Ag presentation was indicated by 2- to 3-fold increase in IFN-gamma production by the T cell clone, as compared with the ALVAC-infected DC alone. Cocultures of ALVAC MART-1-infected and uninfected DC are able to induce MART-1-specific T cell immune responses, as assessed by HLA class I/peptide tetramer binding, IFN-gamma ELISPOT assays, and cytotoxicity tests.
[0192]The amino acid sequence of melanoma antigen MART-1 as given in GenBank as 1082589|pir∥A55253 melanoma antigen MART-1--human is presented in Table 15.1. Predicted MHC Class II-presented epitopes of MART-1/Melan-A are listed in Table 15.2. Experimentally defined MHC Class II-presented epitopes of MART-1/Melan-A are listed in Table 15.3. Designed Ii-Key/MART-1/Melan-1 hybrids containing some of the MHC Class II-presented epitopes of Tables 15.2 and 15.3 are listed in Table 15.4. Predicted MHC Class I-presented epitopes of MART-1/Melan-A are listed in Table 15.5. Experimentally defined MHC Class I-presented epitopes of MART-1/Melan-A are listed in Table 15.6. Designed Ii-Key/MART-1 hybrids containing some of the MHC Class I- and Class II-presented epitopes of Tables 15.2, 15.3, 15.5, and 15.6 are listed in Table 15.7.
TABLE-US-00083 TABLE 15.1 Deduced amino acid sequence of melanoma antigen MART-1. (SEQ ID NO: 531) 1 mpredahfiy gypkkghghs yttaeeaagi giltvilgvl lligcwycrr 51 rngyralmdk slhvgtqcal trrcpqegfd hrdskvslqe kncepvvpna 101 ppayeklsae qspppysp
TABLE-US-00084 TABLE 15.2 Predicted MHC Class II-presented epitopes of MART-1/Melan-A. PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO: 15.2.1 33 LTVILGVLL 5.00 -- 532 15.2.2 35 VILGVLLLI 4.70 -- 533 15.2.3 96 VVPNAPPAY 4.10 6 534 15.2.4 30 IGILTVILG 4.28 -- 535 15.2.5 9 IYGYPKKGH 4.10-5.10 -- 536
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00085 TABLE 15.3 Experimentally defined MHC Class II-presented epitopes of MART-1/Melan-A. PEPTIDE Pos. Sequence Ii-Key SEQ ID NO: 15.3.1 51 RNGYRALMDKSLHVGTQ -- 537 CALTRR
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Peptide 15.3.1 is presented by HLA-DR4 (Zarour H. Proc Natl Acad Sci USA. 2000 97:400-5).
TABLE-US-00086 TABLE 15.4 Designed Ii-Key/MART-1/Melan-1 hybrids containing some of the MHC Class II-presented epitopes of Table 1. SEQ ID PEPTIDE Pos. Sequence NO: A. Non-overlapping 15.4.1 95 Ac-LRMK-ava- 538 VVPNAPPAY-NH2 15.4.2 8 Ac-LRMK-ava- 539 IYGYPKKGH-NH2 15.4.3 51 Ac-LRMK-ava- 540 RNGYRALMDKSLHVGTQCAL TRR-NH2 B. Overlapping 15.4.4 32 Ac-LRMK-ava-LTVILGVLL-NH2 541 15.4.5 34 Ac-LRMK-ava-VILGVLLLI-NH2 542 15.4.6 29 Ac-LRMK-ava-IGILTVILG-NH2 543 15.4.7 29/32/34 Ac-LRMK-ava-IGILTVILGVLLLI-NH2 544
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 15.2.
TABLE-US-00087 TABLE 15.5 Predicted MHC Class I-presented epitopes of MART-1/Melan-A. PEPTIDE Pos. Sequence Score SEQ ID NO: 15.5.1 40 LLLIGCWYC 1289.01 545 15.5.2 56 ALMDKSLHV 1055.10 546 15.5.3 25 EEAAGIGIL 40.0 547 15.5.4 109 AEQSPPPYS 12.0 548
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2. The MHC Class I-presented epitopes of Table 9.4 were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptide 15.5.1 is presented by HLA-A*0201, HLA-A3, and HLA-A31. Peptide 15.5.2 is presented by HLA-A*0201. Peptides 15.5.3 and 15.5.4 are presented by HLA-B40.
TABLE-US-00088 TABLE 15.6 Experimentally defined MHC Class I-presented epitopes of MART-1/Melan-A. PEPTIDE Pos. Sequence SEQ ID NO: 15.6.1 27 AAGIGILTV 549 15.6.2 32 ILTVILGVL 550 15.6.3 24 AEEAAGIGILT 551
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Peptide 15.6.1 is presented by HLA-A*0201 (Kawakami, Y. J Exp Med. 1994 180:347-52). Peptide 15.6.2 is presented by HLA-A*0201 (Castelli C. J Exp Med. 1995 181:63-8). Peptide 15.6.3 is presented by HLA-B*4501 (Schneider J. Intl J Cancer. 1998 75:451-8).
TABLE-US-00089 TABLE 15.7 Designed Ii-Key/MART-1 hybrids containing some of the MHC Class I- and Class II-presented epitopes of 15.2, 15.3, 15.5, and 15.6). SEQ ID NO: Pos. Sequence SEQ ID NO: 15.7.1 27 Ac-LRMK-ava- 552 and AAGIGILTV- 51 RNGYRALMDKSLHVGTQCALT RR-NH2
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II-presented epitope of Table 15.2 and a MHC Class I-presented epitope of Table 15.6.
Example 16
Ii-Key/Her-2 Neu Antigenic Epitope Hybrids
[0193]Immunotherapy directed against the epidermal growth factor receptor which is overexpressed on some cancer cells can control the growth of those tumors. HER-2/neu is over-expressed on tumors in up to 30% of patients with invasive breast cancer and that over-expression is associated with poor clinical outcome. Carr et al. demonstrated in a retrospective consecutive series from 1995 to 1999 that the HER-2/neu gene was amplified in invasive breast carcinomas of 40 of 90 patients (43%) (Carr J A. Arch Surg. 2000 135:1469-7420). Following initial therapy, patients with HER-2/neu amplification had a shorter median disease-free interval (22 months) than did patients with breast cancers not amplifying that gene (40 months; p=0.003). Disease recurred in seventy-two (72%) patients, with 18 (25%) recurring locally. HER-2/neu gene amplification is an independent prognostic indicator for a subset of breast cancer patients who are at high risk for early recurrence regardless of tumor grade, estrogen/progesterone receptor status, and lymph node status. In both early stage, lymph node-negative and node-positive disease, as well as in women with metastatic disease, HER-2/neu overexpression is associated with worse survival. Women with tumors that overexpress HER-2/neu have a less favorable outcome despite adjuvant treatment with either hormonal therapy or chemotherapy. Among HER-2/neu-negative, early stage patients in the Naples GUN trial, tamoxifen benefited overall survival. However, among patients with HER-2/neu-gene amplification, tamoxifen did not improve survival (De Placido S. Br J Cancer. 1990 62:643-6). HER-2/neu over-expression is an independent predictor for tamoxifen failure. Over-expression of HER-2/neu is selective for tumor cells and is observed early in the course of malignant transformation. More importantly, the cytological characteristics of HER-2/neu over-expression (32%) in primary and metastatic lesions is nearly identical (Masood S. Ann Clin Lab Sci. 2000 30:259-65). Inasmuch as micrometastases are the primary source of relapse following primary therapy and HER-2/neu is over-expressed in metastases, HER-2/neu is an excellent target for immunotherapy of patients with early disease, both to consolidate the anti-tumor response locally and to eradicate micrometastases. Likewise, HER-2/neu should be targeted in conjunction with other major treatment regimens in patients who have relapsed following initial therapy.
[0194]Of many approaches to targeting HER-2/neu, the clinically most advanced approach is passive immunotherapy with trastuzumab (Herceptin®), an FDA-approved humanized monoclonal antibody that binds to the extracellular domain of the HER-2/neu receptor for epidermal growth factor (EGF). This monoclonal antibody is indicated both as a single agent and in combination with classical chemotherapies. Slamon et al. evaluated Herceptin® in combination with doxorubicin and cyclophosphamide (AC), or paclitaxel in 496 women with metastatic breast carcinomas that over expressed HER-2/neu (Vogel C L. J Clin Oncol. 2002 20:719-26; Slamon D J. N Engl J Med. 2001 344:783-92). Patients receiving Herceptin®, as compared to patients randomized to chemotherapy alone (either paclitaxel or AC), had a significantly longer time to disease progression (7.4 mo vs. 4.6 mo; p<0.0001), a higher rate of objective response (50% vs. 32%; p<0.001), a longer duration of response (median 9.1 vs. 6.1; p<0.001), a higher 1 year survival rate (78% vs. 67%; p=0.008), longer survival (median survival 25.1 mo vs. 20.3 mo; p=0.046), and a 20% reduction in the risk of death.
[0195]While clinical trials might proceed to alternate trastuzumab dosing regimens and combination therapies, one can suggest that the mechanism of action of trastuzumab will not lead to significantly increased efficacy. Specifically, Trastuzumab blocks the HER-2/neu EGF receptor and induces antibody dependent cellular cytotoxicity (Sliwkowski M X. Semin Oncol. 1999 4 Suppl 12:60-70). ADCC does not lead to antigen-specific memory of T- or B-lymphocytes, nor does it induce proliferation of antigen-specific cytotoxic T-lymphocytes.
[0196]HER-2/neu is also the target for several vaccine trials to induce an active specific immune response. In the NCI PDQ, three current clinical trials use HER-2/neu protein, antigen-pulsed dendritic cells, liposome-encapsulated HER-2/neu MHC peptide epitopes, and a DNA vaccine (http://www.cancer.gov/cancer_information/doc.aspx?viewid=F2 AFAEA4-64BD-4E44-B421-56026E252389). The rationale, of course, is to enhance therapeutic efficacy and clinical ease of administration by inducing: (1) antigen-specific CD8.sup.+ and CD4.sup.+ lymphocytes; (2) autoantibodies against HER-2/neu with memory B-cells; and (3) memory helper T cells.
[0197]Compared to cell-based vaccines, DNA vaccines, and gene therapy approaches, peptide vaccination is preferred for several reasons. Specifically, peptide vaccines are: (1) easily constructed and manufactured; (2) chemically stable; (3) free of adventitious agents and other pathogens; and, (4) devoid of oncogenic potential. Until recently, most groups have focused on the use of MHC Class I peptide vaccines, which have triggered low-intensity CD8+ cytotoxic T cell responses. Shiku and colleagues have identified a novel human Her-2/neu2-derived peptide which is homologous to a mouse H-2Kd-restricted tumor antigen induces HLA-A24-restricted cytotoxic T lymphocytes in ovarian cancer patients and healthy individuals (Okugawa T. Eur J Immunol. 2000 30:3338-46; Ikuta Y. Int J Cancer. 2000 87: 553-8; Nagata Y. J Immunol. 1997 159:1336-43). In addition they have demonstrated presentation of a MHC Class I-binding peptide by monocyte-derived dendritic cells incorporating a hydrophobized polysaccharide-truncated Her-2/neu protein complex (Ikuta Y. Blood. 2002 99:3717-24; Araki H. Br J Haematol. 2001 114:681-9).
[0198]Peptide vaccines do enhance responses by CTL cells recognizing MHC Class I-presented peptides, but can be augmented by also immunizing T helper cells with MHC Class II-presented peptides. HER-2/neu-derived, MHC Class II-presented peptides are expressed by human breast, colorectal and pancreatic adenocarcinomas and are recognized by in vitro-induced, specific CD4.sup.+ T cell clones (Perez S. Cancer Immunol Immunother. 2002 50:615-24; Sotiriadou R. Br J Cancer. 2001 85:1527-34). Murray et al. showed that the Her-2/neu(777-789) peptide induced peripheral blood mononuclear cells from patients with metastatic breast cancer to secrete IFN-γ (Murray J L. Semin Oncol. 2000 27 Suppl:71-5). This group also showed that Her-2/neu(369-377) induced strong CTL response in peripheral blood mononuclear cells from healthy donors (Anderson B W. Clin Cancer Res. 2000 6:4192-200; Anderson B W. Cancer Immunol Immunother. 2000 49:459-68), as well as the secretion of CXC chemokine IP-10 from peripheral blood mononuclear cells from breast cancer patients and healthy donors (Lee T V. J Interferon Cytokine Res. 2000 20:391-401). However, in a clinical trial with that MHC Class I peptide only 3/9 patients had lymphocyte proliferative responses that were above baseline following vaccination (Murray J L. Semin Oncol. 2000 27 Suppl:71-5). Increased CTL proliferation and IFN-a levels were seen in stimulated cultures of peripheral blood mononuclear cells of only one vaccinated patient. In 3 of 5 patients, IFN-a and CTL activity were increased significantly by IL-12 addition, indicating that weak antigen presentation leads to weak CTL induction, which is reversed partially in vitro with pro-inflammatory cytokines. However, MHC Class I peptide immunization does not induce helper CD4.sup.+ T cell responses. For this reason, peptide vaccines are sought with either only a MHC Class II presented, CD4.sup.+ T-helper cell stimulating epitope or with a peptide in which a MHC Class II-presented, CD4+ T-helper cell stimulating epitope overlays a MHC Class I-presented, CD8.sup.+ T-cytotoxic cell stimulating epitope.
[0199]Peripheral blood mononuclear cells from healthy donors and ovarian cancer patients do respond to Her-2/neu peptides (Fisk B. Anticancer Res. 1997 17:45-53). Peptide sequences from Her-2/neu containing anchors for major human MHC-class II molecules induced proliferative and cytokine responses at a higher frequency in healthy donors than in ovarian cancer patients. Four Her-2/neu peptides of sequences: 396-406, 474-487, 777-789, and 884-899 stimulated proliferation of a larger number of healthy donors than three other distinct HER-2 peptides 449-464, 975-987 and 1086-1098. The pattern of responses of twenty-five ovarian cancer patients was different from that of healthy donors. T cell lines were developed by stimulation with peptides of peripheral blood mononuclear cells of an ovarian cancer patient who showed a stable response to all four Her-2/neu peptides over six months. Each T cell line differed in secretion of IFN-gamma and IL-10. These results demonstrate (a) that Her-2/neu peptides can stimulate expansion of T cells in both healthy donors and ovarian cancer patients, and (b) different peptides induce different cytokine secretion patterns. (J Interferon Cytokine Res. 2002 May; 22(5):583-92)
[0200]Ioannides and colleagues demonstrated axillary lymph nodes from patients with breast carcinoma respond to HER-2/neu peptides (Kuerer H M. J Interferon Cytokine Res. 2002 22:583-92). Freshly isolated lymphocytes from lymph nodes of 7 women undergoing surgery for invasive breast cancer were stimulated with HER-2/neu peptides at 50 μm/ml and with control antigens. IFN-γ, IL-4, and IL-10 levels were determined at priming and at restimulation with HER-2/neu peptides. Lymphocytes isolated from the axillary lymph nodes of the patients responded to HER-2/neu peptides, proliferating and specific cytokine production. Proliferative responses to HER-2/neu peptides were seen in lymphocytes of patients with and without overexpression of HER-2/neu in the primary tumor. In some patients, the proliferative response to HER-2/neu peptides in lymphocytes from lymph nodes with metastases was absent or decreased compared to response in lymphocytes from lymph nodes without metastases from the same patient (p<0.05). HER-2/neu peptides induced a predominantly T helper type 1 (Th1) pattern of cytokine response in nodal lymphocytes isolated from breast cancer patients. A Th1-specific cytokine production pattern was maintained at priming and restimulation with HER-2/neu peptides and was amplified with IL-12 costimulation. These results indicate that HER-2/neu peptides can activate T cells in draining lymph nodes from women with invasive breast cancer.
[0201]Patients immunized with an HLA-A2-presented, Her-2/neu peptide developed only a low level and short-lived CTL response, in the absence of concurrent vaccination with a MHC Class II-presented epitope (Ward R L. Hum Immunol. 1999 60:510-5). Six HLA-A2 patients with Her-2/neu-overexpressing cancers received 6 monthly vaccinations with a vaccine preparation consisting of 500 μg of Her-2/neu(369-377) peptide, admixed with 100 μg of GM-CSF. The patients had either stage III or IV breast or ovarian cancer. Immune responses to the Her-2/neu(369-377) peptide were examined using an IFN-γ enzyme-linked immunosorbent spot assay. Although HER-2/neu MHC class I epitopes induced HER-2/neu peptide-specific IFN-γ-producing CD8+ T cells, the magnitudes of the responses were low, as well as short-lived, indicating that CD4+ T-cell help is required for robust and lasting immunity to this epitope.
[0202]Disis and colleagues immunized with breast cancer patients a HER-2/neu helper peptide vaccine generating HER-2/neu CD8 T-cell immunity (Knutson K L. J Clin Invest. 2001 107:477-84). Nineteen HLA-A2 patients with HER-2/neu-overexpressing cancers received a vaccine preparation consisting of Her-2/neu(369-384), Her-2/neu(688-703), and Her-2/neu(971-984). Contained within these sequences are HLA-A2-binding motifs Her-2/neu(369-377), Her-2/neu(689-697), and Her-2/neu(971-979). After vaccination, the mean peptide-specific T-cell precursor frequency to the HLA-A2 peptides increased in the majority of patients. In addition, the peptide-specific T cells were able to lyse tumors. The responses were long-lived and detected for more than 1 year after the final vaccination in some patients. These results demonstrate that Her-2/neu MHC class II epitopes containing overlaying MHC Class I epitopes induce long-lasting Her-2/neu-specific IFN-γ-producing CD8.sup.+ T cells.
[0203]Disis and colleagues immunized sixty-four patients with HER-2/neu-overexpressing breast, ovarian, or non-small-cell lung cancers with vaccines composed of peptides derived from potential T-helper epitopes of the HER-2/neu protein mixed with granulocyte-macrophage colony-stimulating factor and administered intradermally (Disis M L. J Clin Oncol. 2002 20:2624-32). Nine different epitopes were used: 3 derived from the intracellular domain of her-2/neu (p776-790, p927-941, and p1166-1180), 3 derived from the extracellular domain of her-2/neu (p42-56, p98-114, and p328-345), and 3 with helper epitopes that encompass in their natural sequence HLA-A2 binding motifs (p369-384, p688-703, and p971-984). Ninety-two percent of patients developed T-cell immunity to HER-2/neu peptides and 68% to a HER-2/neu protein domain. Epitope spreading was observed in 84% of patients and correlated with the generation of a HER-2/neu protein-specific T-cell immunity (P=0.03). At 1-year follow-up, immunity to the HER-2/neu protein persisted in 38% of patients. No patient developed any detected autoimmune toxicity, particularly in organs known to express basal levels of her-2/neu protein including the liver, digestive tract, and skin. The incorporation of MHC Class II epitopes used in this study in Ii-Key hybrid molecules might lead to more rapid anti-her-2/neu immune responses with lower and fewer doses, greater epitope spreading, induction of higher affinity T-cells against tumor, more prolonged immune responses against epitopes and her-2/neu protein, and greater clinical efficacy.
[0204]Finding tumor-reactive CTLs in tumor infiltrates and in the peripheral blood of cancer patients, raises the question that any anti-tumor immune response does not control disease spread (Anderson B W. Clin Cancer Res. 2000 6:4192-200). One might then question whether amplification of this response by peptide vaccines is useful during disease progression. Induction of tumor-reactive CTLs in healthy donors at risk, as well as in patients free of disease, has been proposed on the hypothesis that CTLs that recognize tumors early are more effective in containing their progression than CTLs that expand only when the disease progresses. Priming of cytolytic T cell activity in 10 healthy donors was tested with Her-2/neu(369-377) peptide as an immunogen and autologous peripheral blood mononuclear cell-derived dendritic cells as antigen presenting cells. Of those two responded at priming with Her-2/neu(369-377) peptide presented on autologous dendritic cells by induction of Her-2/neu(369-377) peptide-specific CTL activity. Three other responders were identified after two additional restimulations. Induction of cytolytic activity at priming was enhanced in responders by tumor necrosis factor-alpha and IL-12 but not in the non-responders.
[0205]Determinant spreading and Th1 responses were induced by in vitro stimulation with Her-2/neu peptides (Anderson B W. Cancer Immunol Immunother 2000 49:459-68). The induction of a response to Her-2/neu(776-789) induced reactivity to other Her-2/neu peptides. Her-2/neu(776-789) expanded a response to Her-2/neu (884-899) in both an ovarian cancer patient with progressive disease and a healthy donor who shared HLA-DR11. This response was characterized mainly by increased IFN-γ secretion, and proliferation, but did not occur with another donor who shared only HLA-DR14 and HLA-DQ5 with the patient. Epitope spreading can also be enhanced by the coordinated use of Ii-Key/antigenic epitope hybrids immunizations with Ii reverse gene construct, Her-2/neu gene immunizations.
[0206]Hess and colleagues found that a chimeric construct of an MHC class II binding peptide from Her-2/neu and the N-terminal flanking region of CLIP elicited potent antitumor activity against a Her-2/neu-positive tumor in a rat model system (Hess A D. Clin Immunol 2001 101:67-76). Induction of effective antitumor immunity required presentation of the chimeric peptide on irradiated tumor cells or in concert with a Her-2/neu MHC class I-restricted peptide from Her-2/neu. Adoptive transfer studies showed the need for CD4 T helper cells for protective antitumor immunity. Immunization with the epitope-only peptide caused a weak immune response to the unmodified peptide in vitro of both type 1 (IL-2, IFN-γ) and type 2 (IL-4, IL-10) cytokine-producing cells analyzed by RT-PCR (qualitative and quantitative) and by limiting dilution assay. Comparatively, immunization with the chimeric construct elicited a potent immune response to the parent epitope with predominantly type 1 cytokine-producing cells.
[0207]Accelerated Her-2/neu degradation enhanced ovarian tumor recognition by CTL (Castilleja A. Mol Cell Biochem. 2001 217:21-33). In those studies, Her-2/neu degradation was enhanced in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed Her-2/neu by the addition of geldanamycin, which down-modulated Her-2/neu from the cell surface and promoted its polyubiquitinylation and degradation. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, Her-2/neu(369-377) from SKOV.A2 was inhibited by proteosome inhibitors, such as LLnL. Additional experiments indicated that the newly synthesized Her-2/neu in the presence of GA was the main source of epitopes recognized by CTL. Twenty-hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of Her-2/neu CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells thereby promoting immunogenecity. Similarly geldanamycin and other compounds acting by a similar mechanism, are expected to enhance binding of MHC Class II epitopes in the ER in the absence of Ii protein.
[0208]Ward and colleagues used phage-displayed ErbB-2 gene fragment libraries and synthetic peptides to epitope-map a panel of anti-Her-2/neu monoclonal antibodies (Yip Y L. Cancer Immunol Immunother. 2002 50:569-87; Yip Y L. J Immunol. 2001 166:5271-8). The epitopes of three monoclonal antibodies, N12, N28, and L87, were successfully located to Her-2/neu(C531-A586), Her-2/neu(T216-C235), and Her-2/neu(C220-C235) of Her-2/neu, respectively. It was found that while N12 inhibited tumor cell proliferation, N28 stimulated the proliferation of a subset of breast cancer cell lines over-expressing Her-2/neu. The peptide region recognized by N12, Her-2/neu(C531-A586), was used as an immunogen to selectively induce an inhibitory immune response in mice. Mice immunized with the GST fusion peptide, GST-Her-2/neu(C531-A586), recognized native Her-2/neu, the peptide Her-2/neu(531-586), three 15-amino acid peptides of Her-2/neu(533-548), Her-2/neu(545-5560), and Her-2/neu(571-586). More importantly, immunoglobulins purified from mouse sera were able to inhibit up to 85% of tumor cell proliferation. This study supports the use of some of the potential antibody recognized determinants in the construction of Ii-Key/Her-2/neu MHC Class II-presented antigenic epitope/antibody-recognized determinant hybrids. The antibody recognized determinants are presented in Table 16.8 and hybrids containing those epitopes are presented in Table 16.9. Such hybrids containing antibody-recognized determinants might be preferred can be used for the development of both passive and active immunotherapies of Her-2/neu over-expressing tumors.
[0209]Given the experimentally identified MHC Class II-presented epitopes (above) such epitope can be synthesized within Ii-Key/Her-2/neu antigenic epitope hybrids for stimulation of a diagnostic or therapeutic immune response.
[0210]The amino acid sequence of human Her-2/neu protein [Homo sapiens](gi|19575768|) was obtained from GenBank (Table 16.1). An important consideration in the selection of peptides for cancer immunotherapy is the high degree of sequence homology between Her-2/neu and another member of the subclass I family of growth factor receptor (EGF-r) (Lustgarten J. Hum Immunol. 1997 52:109-18). Unlike Her-2/neu, the EGF-r is widely expressed in the body. Peptide sequences identical between Her-2/neu and the mouse or human EGF-r were not selected for two reasons. First, it is likely that T-cell tolerance to such sequences would have eliminated from the repertoire high affinity T cells with specificity for such epitopes. Second, it would be undesirable to target CTL against normal cell expressing EGF-r peptides. Predicted MHC Class II-presented epitopes of Her-2/neu protein are presented in Table 16.2. Experimentally determined MHC Class II-restricted epitope of human Her-2/neu protein are listed in Table 16.3. Designed Ii-Key/Her-2/neu hybrids using some of the MHC Class II-presented epitopes of Tables 2 and 3 are listed in Table 16.4. Predicted MHC Class I-presented epitopes of Her-2/neu protein are listed in Table 16.5. Experimentally determined MHC Class I-presented epitopes of Her-2/neu protein are listed in Table 16.6. Designed Ii-key/MHC Class II epitope/MHC Class I epitope hybrids are listed in Table 16.7. Antibody-recognized determinants on Her-2/neu are listed in Table 16.8 Designed Ii-Key/Her-2/neu hybrids using some of the antibody-recognized determinants of Table 16.8 and MHC Class II-presented epitopes of Tables 2 and 3 are presented in Table 16.9.
TABLE-US-00090 TABLE 16.1 Deduced amino acid sequence of Her-2/neu. 1 melaalcrwg lllallppga astqvctgtd mklrlpaspe thldmlrhly (SEQ ID NO: 553) 51 qgcqvvqgnl eltylptnas lsflqdiqev qgyvliahnq vrqvplqrlr 101 ivrgtqlfed nyalavldng dplnnttpvt gaspgglrel qlrslteilk 151 ggvliqrnpq lcyqdtilwk difhknnqla ltlidtnrsr achpcspmck 201 gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc aagctgpkhs 251 dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp 301 ynylstdvgs ctlvcplhnq evtaedgtqr cekcskpcar vcyglgmehl 351 revravtsan iqefagckki fgslaflpes fdgdpasnta plqpeqlqvf 401 etleeitgyl yisawpdslp dlsvfqnlqv irgrilhnga ysltlqglgi 451 swlglrslre lgsglalihh nthlcfvhtv pwdqlfrnph qallhtanrp 501 edecvgegla chqlcarghc wgpgptqcvn csqflrgqec veecrvlqgl 551 preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc 601 psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp 651 ltsiisavvg illvvvlgvv fgilikrrqq kirkytmrrl iqetelvepl 701 tpsgampnqa qmrilketel rkvkvlgsga fgtvykgiwi pdgenvkipv 751 aikvlrents pkankeilde ayvmagvgsp yvsrllgicl tstvqlvtql 801 mpygclldhv renrgrlgsq dllnwcmqia kgmsyledvr lvhrdlaarn 851 vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft 901 hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid 951 vymimvkcwm idsecrprfr elvsefsrma rdpqrfvviq nedlgpaspl 1001 dstfyrslle dddmgdlvda eeylvpqqgf fcpdpapgag gmvhhrhrss 1051 strsgggdlt lglepseeea prsplapseg agsdvfdgdl gmgaakglqs 1101 lpthdpsplq rysedptvpl psetdgyvap ltcspqpeyv nqpdvrpqpp 1151 spregplpaa rpagatlerp ktlspgkngv vkdvfafgga venpeyltpq 1201 ggaapqphpp pafspafdnl yywdqdpper gappstfkgt ptaenpeylg 1251 ldvpv
TABLE-US-00091 TABLE 16.2 Predicted MHC Class II-presented epitopes of Her-2/neu protein. Ii- SEQ. ID. PEPTIDE Pos. Sequence Score Key NO. 16.2.1 985 FVVIQNEDL 7.40 6 554 16.2.2 98 LRIVRGTQL 7.30 4 555 16.2.3 952 MIMVKCWMI 7.20 -- 556 16.2.4 894 LRRRFTHQS 7.00 6 557 16.2.5 684 YTMRRLLQE 6.70 6 558 16.2.6 664 VVLGVVFGI 5.90 -- 559 16.2.7 1041 MVHHRHRSS 5.60 -- 560 16.2.8 421 LSVFQNLQV 5.50 -- 561 16.2.9 180 LTLIDTNRS 5.40 4 562 16.2.10 670 FGILIKRRQ 5.40 -- 563 16.2.11 396 LQVFETLEE 5.20 -- 564 16.2.12 61 LTYLPTNAS 5.10 11 565 16.2.13 951 YMIMVKCWM 5.00 -- 566 16.2.14 719 LRKVKVLGS 5.00 4 567 16.2.15 424 FQNLQVIRG 5.20 -- 568
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for high scoring selections with multiple common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00092 TABLE 16.3 Experimentally determined MHC Class II- restricted epitope of human Her-2/neu protein. SEQ. ID. PEPTIDE Pos. Sequence NO. 16.3.1 884 VPIKWMALESILRRR 569 16.3.2 776 GSPYVSRLLGICL 570 16.3.3 396 QLQVFETLEEI 571 16.3.4 474 LCFVHTVPWDQLF 572 16.3.5 450 GISWLGLRSLRE 573 16.3.6 975 EFSRMARDPQRF 574 16.3.7 1086 FDGDLGMAAKGL 575 16.3.8 42 HLDMLRHLYQGCQVV 576 16.3.9 98 LRIVRGTQLFEDNYAL 577 16.3.10 328 TQRCEKCSKPCARVCYGL 578 16.3.11 776 LGSGAFGTVYKGIWI 579 16.3.12 927 PAREIPDLLEKGERL 580 16.3.13 1166 TLERPKTLSPGKNGV 581 16.3.14 369 KKIFGSLAFLPESFDGD 582 16.3.15 688 RQQKIRKYTMRRLLQE 583 16.3.16 971 ELVSEFSRMARDPQ 584
Pos. is the residue position in the primary sequence of the first amino acid in the peptide. Sequence is the amino acid sequence of the experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Peptide 16.3.1 was reported by Perez S. et al. (Cancer Immunol Immunother. 2002 50:615-24). Peptide 16.3.2 was reported by Sotiriadou R. et al. (Br J Cancer. 2001 85:1527-34). Peptide 16.3.3 was reported by Fisk B. et al. (Anticancer Res. 1997 17:45-53). Peptides 16.3.8-16.3.16 are those reported in a Phase I clinical trial by Disis and colleagues (Disis M L. J Clin Oncol 2002 20:2624-32). Peptide 16.3.9 contains a predicted HLA-DRB1-0101-presented motif LRIVRTGTQL (SEQ ID NO: 585) and PEPTIDE 16.3.16 contains a DRB1-0101-presented motif LVSEFSRMA (SEQ ID NO: 586); both stimulated lymphocytes from an immunized patients. Additional peptides in the series studied by Disis et al. might be found to containing MHC Class II-presented motifs when tested for additional HLA-DB alleles and to lower indices for scoring. Such epitopes are subject to being incorporated in Ii-Key/Her-2 antigenic epitope hybrids.
TABLE-US-00093 TABLE 16.4 Designed Ii-Key/Her-2/neu hybrids using some of the MHC Class II-presented epitopes of Tables 2 and 3. SEQ. ID. PEPTIDE Pos. Sequence NO. A. Non-overlapping 16.4.1 776 Ac-LRMK-ava-GSPYVSRLLGICL-NH2 587 16.4.2 396 Ac-LRMK-ava-QLQVFETLEEI-NH2 588 16.4.3 985 Ac-LRMK-ava-FVVIQNEDL-NH2 589 16.4.4 98 Ac-LRMK-ava-LRIVRGTQL-NH2 590 16.4.5 894 Ac-LRMK-ava-LRRRFTHQS-NH2 591 16.4.6 684 Ac-LRNK-ava-YTMRRLLQE-NH2 592 16.4.7 1041 Ac-LRMK-ava-MVHHRHRSS-NH2 593 16.4.8 972 Ac-LRMK-ava-LVSEFSRMA-NH2 594 B. Overlapping 16.4.8 884, Ac-LRMK-ava- 595 894 VPIKWMALESILRRRFTHQS-NH2 16.4.9 664, Ac-LRMK-ava-VVLGVVFGILIKRRQ-NH2 596 670 16.4.10 951, Ac-LRMK-ava-YMIMVKCWMI-NH2 597 952 16.4.11 421, Ac-LRMK-ava-LSVFQNLQVIRG-NH2 598 424
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 16.2 and 16.3.
TABLE-US-00094 TABLE 16.5 Predicted MHC Class I-presented epitopes of Her-2/neu protein. SEQ. ID. PEPTIDE Pos. Sequence Score NO. 16.5.1 661 ILLVVVLGV 1006.2 599 16.5.1 369 KIFGSLAFL 481.2 600 16.5.1 167 ILWKDIFHK 450.0 601 16.5.1 63 TYLPTNASL 360.0 602 16.5.2 106 QLFEDNYAL 324.1 603 16.5.3 553 EYVNARHCL 300.0 604 16.5.4 440 AYSLTLQGL 240.0 605 16.5.5 907 SYGVTVWEL 220.0 606 16.5.6 1022 EYLVPQQGF 180.0 607 16.5.7 689 RLLQETELV 126.1 608 16.5.8 714 ILKETELRK 60.0 609 16.5.9 754 VLRENTSPK 30.0 610 16.5.10 673 ILIKRRQQK 30.0 611
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. The MHC Class I-presented epitopes were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90).
TABLE-US-00095 TABLE 16.6 Experimentally determined MHC Class I-presented epitopes of Her-2/neu protein. SEQ. ID. PEPTIDE Pos. Sequence NO. 16.6.1 106 QLFEDNYAL 612 16.6.2 369 KIFGSLAFL 613 16.6.3 689 RLLQETELV 614 16.6.4 435 ILHNGAYSL 615 16.6.5 665 VVLGVVFGI 616 16.6.6 952 YMIMVKCWM 617 16.6.7 654 IISAVVGIL 618 16.6.8 654 FLSAVVGILV 619 16.6.9 773 VMAGVGSPYV 620 16.6.10 754 VLRENTSPK 621
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class I-presented epitope. Peptide 16.6.1 is presented by HLA-A2.1 (Kono K. Int j Cancer. 1998 78:202-8). Peptide 16.6.2 is presented by HLA-A2.1 (Kono K. Int J Cancer. 1998 78:202-8), as confirmed by Rongcun Y., et al. (J Immunol. 1999 163:1037-44). It was also shown to be immunogenic in double transgenic mice expressing HLA-A2.1 and human CD8 (Lustgarten J. Hum Immunol. 1997 52:109-18). Peptide 16.6.3 is presented by HLA-A2.1 (Kono, K. Int J Cancer. 1998 78:202-8; Rongcun Y. J Immunol. 1999 163:1037-44). It was nonimmunogenic in the study of Lustgarten J. et al. (Hum Immunol. 1997 52:109-18). Peptides 16.6.4, 16.6.5 and 16.6.6 are presented by HLA-A2.1 (Rongcun Y. J Immunol. 1999 163:1037-44). Peptide 16.6.7 is presented by HLA-A2 (Peoples G. Proc Natl Acad Sci USA. 1995 92:432-6) and is nonimmunogenic in the study of Lustgarten, J. et al. (Hum Immunol. 1997 52:109-18). Peptide 16.6.8 is presented by HLA-A2 (Tanaka Y. Int J Cancer. 2001 94:540-4). Peptide 16.6.9 is presented by HLA-A2.1 (Lustgarten J. Hum Immunol. 1997 52:109-18). Peptide 16.6.10 is presented by HLA-A3 (Kawashima I. Cancer Res. 1999 59:431-5).
TABLE-US-00096 TABLE 16.7 Designed Ii-key/MHC Class II epitope/MHC Class I epitope hybrids. SEQ. ID. PEPTIDE Pos. Sequence NO. 16.7.1 II: 76, Ac-LRMK-ava- 622 I: 73 VMAGVGSPYVSRLLGICL-NH2 16.7.2 II: 396, Ac-LRMK-ava-QLQVFETLEEI- 623 I: 369 KIFGSLAFL-NH2 16.7.3 II: 670, Ac-LRMK-ava-FGILIKRRQQK-NH2 624 I: 673
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope, with MHC Class II indicated as I: and MHC Class II indicated as II:. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2.
TABLE-US-00097 TABLE 16.8 Antibody-recognized determinants on Her-2/neu. Peptide Pos. Sequence SEQ ID NO: 16.8.1 216 TRTVCAGGCARCKGP 625 16.8.2 220 CAGGCARCKGPLPTD 626 16.8.3 533 QFLRQECVEECRVLQ 627 16.8.4 545 VLQGLPREYVNARHC 628 16.8.5 571 NGSVTCFGPEADQCV 629
These peptides are reported to react with serums of mice which were immunized with a GST fusion protein containing the Her-2/neu(C220-C235) sequence (Yip Y L. Cancer Immunol Immunother. 2002 50:569-87; Yip Y L. J Immunol. 2001 166:5271-8).
TABLE-US-00098 TABLE 16.9 Designed Ii-Key/Her-2/neu hybrids using some of the antibody-recognized determinants of Table 16.8 and MHC Class II-presented epitopes of Tables 2 and 3. SEQ. ID. PEPTIDE Pos. Sequence NO. A. Non-overlapping (MHC Class II and antibody- recognized epitopes) 16.9.1 776; Ac-LRMK-ava-GSPYVSRLLGICL- 630 216, TRTVCAGGCARCKGPLPTD-NH2 220 16.9.2 396; Ac-LRMK-ava-QLQVFETLEEI- 631 571 NGSVTCFGPEADQCV-NH2 B. Overlapping 16.9.3 534; Ac-LRMK-ava-SQFLRGQECVEECRVLQ- 632 533 NH2 16.9.4 555; Ac-LRMK-ava-RVLQGLPREYVNARHC- 633 556 NH2
Pos. is the residue position in the primary sequence of the first amino acid in the MHC Class II-presented epitope and after the semicolon is the first residue in the peptide reported to contain an antibody-recognized epitope. Sequence is the amino acid sequence of a hybrid peptide.
Example 17
Ii-Key/Anthrax MHC Class II Antigenic Epitope Hybrids
[0211]Ii-Key/antigenic epitope hybrids can be applied as vaccines against anthrax and other bioterrorism agents. In order to understand well the applications of Ii-Key/antigenic epitope hybrids as stand-alone vaccines or as components of a multivaccine protocol against anthrax, a review of the biology and pathogenesis of bacillus anthracis is useful. Likewise, the currently available vaccines against anthrax are considered in light of improvements offered by the products and methods of this disclosure. Specifically, the Ii-Key/antigenic epitope hybrid technology provides for enhanced antigen-specific T-helper cell responses, which enable existing vaccines and independently offer a significant degree of protection against anthrax infection. The Ii-Key/anthrax epitope peptide vaccine offers safety and effectiveness for use by both military and civilian populations.
[0212]Anthrax is an infectious disease caused by the spores of the bacterium, Bacillus anthracis, a large gram-positive, non-motile, bacterial rod. Human anthrax disease has three major forms: cutaneous, inhalational, and gastrointestinal. If untreated, anthrax in all forms can lead to septicemia and death. Early treatment of cutaneous anthrax is usually curative. Patients with gastrointestinal anthrax have reported case fatalities of 25% to 75%. Case fatality rates for inhalation anthrax are 90% to 100%. Early treatment of all forms of anthrax with antibiotics is essential because antibiotics are ineffective once the bacteria grow densely enough to secrete anthrax toxin (Leppla S H. Nature Medicine. 2001 7:659-660). Inhalational anthrax has two phases. During the first phase, which occurs within one to five days following exposure, the patient has flu-like symptoms (cough, malaise, fatigue and mild fever). The following phase includes sudden onset of severe respiratory distress, chest pain, and fever. Within a day, septic shock and death will likely occur. In the case of inhalational anthrax, antibiotic therapy is of limited benefit except when given immediately following exposure.
[0213]Anthrax toxin, the major virulence factor produced by B. anthracis, consists of three proteins. PA binds to human cells and forms a channel through which LF, the dominant virulence factor, enters the cytosol (Leppla S H. Nature Medicine. 2001 7:659-660). LF is a metalloproteinase that cleaves mitogen-activated protein kinases (MEKs), resulting in cell death and a clinical picture resembling septic shock. It is the binding of LF to PA63 (an area on PA that is made available following cellular binding and furin catalysis of PA) that triggers LF-PA63 binding, oligomerization, heptamer formation, and cytosolic transport of LN (Leppla S H. Bacterial protein toxins (eds. Fehrenbach F. et al.) 111-112 Gustav Fischer, N.Y., 1988).
[0214]Anthrax lethal toxin comprises two proteins: protective antigen (PA; MW 83 kDa) and lethal factor (LF; MW 87 kDa). The crystal structure of PA was determined in monomeric and heptameric forms (Liddington R. J Appl Microbiol. 1999 87:282-290). It bears no resemblance to other bacterial toxins of known three-dimensional structure, and defines a new structural class, which includes homologous toxins from other Gram-positive bacteria. Membrane insertion involves the water-soluble heptamer undergoing a substantial pH-induced conformational change thereby creating a 14-stranded beta-barrel. Recent work by Collier's group lends support to this model of membrane insertion (Benson E L. Biochemistry. 1998 37:3941-8). Lethal factor is the catalytic component of anthrax lethal toxin. It binds to the surface of the cell-bound PA heptamer and, following endocytosis and acidification of the endosome, translocates to the cytosol.
[0215]Liddington and colleagues determined the crystal structure of the anthrax lethal factor (Pannifer A D. Nature 2001 414:229-33). Lethal factor (LF) is highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near their amino termini, leading to the inhibition of one or more signaling pathways. The crystal structure of LF and its complex with the N terminus of MAPKK-2 was determined. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic center; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.
[0216]Proteasome activity is required for anthrax lethal toxin to kill macrophages (Tang G. Infect Immun. 1999 67:3055-60). Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines. LF is translocated by PA into the cytosol of target cells, where it cleaves mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins. Proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not. Various data indicate that the proteasome mediates a toxic process initiated by LF in the cell cytosol. This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis. Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates.
[0217]Leppla and colleagues found oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells (Singh Y. Infect Immun. 1999 67:1853-9). The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule.
[0218]Leppla and colleagues identified three advances which is point to possible therapies by inhibiting the toxin (Chaudry G J. Trends Microbiol. 2002 10:58-62). Identification of the cell surface toxin receptor could lead to the design of binding competitors and receptor decoys. Determination of the crystal structure of the lethal factor protease will facilitate ongoing efforts to develop protease inhibitors as therapies. Finally, the susceptibility of certain inbred mice to anthrax lethal toxin was associated with mutations in the kinesin-like protein Kif1C, a discovery that could help to explain how anthrax toxin kills animals.
[0219]Various vaccine strategies have been developed to protect humans against the pathological effects of PA and LF, which are released by Bacillus anthracis. In order to appreciate the usefulness of Ii-Key(MHC Class II and Ii-Key/MHC Class II epitope/ARD hybrids in augmenting those vaccines, it is useful to review the current state of vaccination against and treatment of anthrax infections.
[0220]During the bioterrorism attacks of late 2001 in which thousands of people were potentially exposed to anthrax spores contained in letters to elected officials and employees of media outlets, more than 30,000 individuals received prophylactic antibiotic therapy (principally ciprofloxacin and doxycycline) for 60 days. Because anthrax spores can persist in the lungs of animals for more than 60 days, these potentially exposed individuals were then offered another 40-day course of antibiotics and therapeutic vaccination with the Anthrax Vaccine Adsorbed upon completion of the initial antibiotic regimen. However, since there is only a narrow time-window for effective antibiotic therapy following exposure to anthrax spores, using antibiotics on a mass scale is not a realistic option. Prophylactic and therapeutic vaccination against anthrax and anthrax toxin is thus the most promising form of mass intervention in case of an anthrax-bioterrorism event.
[0221]Prior to its emergence as a potentially preferred bioterrorism weapon, anthrax infection was limited to animals and humans with occupations involving direct and extensive handling of animals or animal products. Approval of the current anthrax vaccine, Anthrax Vaccine Adsorbed was based on a clinical trial conducted by Philip S. Brachman in the 1950's involving U.S. mill workers who processed animal hides. Prior to the availability of this vaccine, the yearly average number of human anthrax cases was 1.2 per 100 employees in these mills. The Brachman study provided evidence for the efficacy of anthrax vaccination: (a) 26 patients developed anthrax during the study--5 inhalation and 21 cutaneous; (b) of the 5 inhalation anthrax cases, 2 patients received placebo and 3 were in the observation group; (c) four of the 5 patients with inhalation anthrax died; (d) of the 21 cases of cutaneous anthrax, 15 individuals received placebo, three were in the observation group, and two individuals were partially immunized, and one individual was fully immunized; (e) the authors calculated vaccine efficacy level of 92.5% for fully vaccinated individuals (Brachman P S. American Journal of Public Health. 1962 52:432-440).
[0222]In 1966 the CDC initiated a clinical study of a vaccine that was a modification of the vaccine used in the Brachman trial. Although both vaccines were based on immunity induced by protective antigen (PA), their methods of preparation differed. The IND trial used three lots of material produced by the Michigan Department of Public Health (MDPH). The data submitted to the Division of Biologics Standards described the CDC's experience with 16,000 doses of the anthrax vaccine administered to 7,000 study participants. Mild local reactions ranged between 3 to 36%, moderate reactions between 1 to 3%, and severe local reactions in less than 1%. Systemic reactions were reported in 4 cases over the 5-year period; these reactions included transient fever, chills, nausea, and general body aches. The vaccine was approved in 1970 for individuals who might contact animal products that might be contaminated with B. anthracis spores, individuals at high risk (including veterinarians), and those engaged in diagnostic or investigational activities that might bring them in contact with the spores.
[0223]In 1985 an Advisory Panel Review under the Public Health Service Act designated the anthrax vaccine produced by MDPH as a Category I product, that is safe, effective and not misbranded (Federal Register 1985 50:51002). The efficacy data from the Brachman study and the safety data from the CDC study were the basis for these findings. In May 1988, the Department of Defense (DOD) approved the prophylactic vaccination of US military personnel. In December 2001, therapeutic vaccination was also initiated in individuals previously exposed to anthrax spores (as a result of acts of bioterrorism in Florida, New York, and Washington, D.C.), and who were receiving prophylactic antibiotic therapy.
[0224]The current AVA vaccine produced by BioPort (the successor to MDPH in anthrax vaccine manufacturing) is derived from a strain of B. anthracis that does not cause anthrax disease. It is a cell-free filtrate containing no whole bacteria. The vaccination protocol includes an initial dose of 0.5 ml s.c., followed by 0.5 ml s.c. booster doses at 2 and 4 weeks, and 6, 12 and 18 months, with yearly boosters thereafter. The manufacturing process is difficult, costly, time consuming, limited in scale, and laden with many biologics controls. Development of a nontoxinogenic and nonencapsulated recombinant B. anthracis spore vaccine and lethal factor DNA vaccine have been initiated recently (Cohen S. Infect Immun. 2000 68:4549-58; Price B M. Infect Immun. 2002 69:4509-15). Also three new anthrax vaccines based on the PA protein are being studied (Friedlander A M. JAMA 1999 282:2104-6; Thomas L J. 4th International Conference on Anthrax. Abstracts Book. Jun. 10-13, 2001, Annapolis, Md., USA; Turnbull P C B. Curr Opin Infect Dis. 2001 13:11). Being products of biologic manufacturing, the process and controls are much more involved and wrought with regulatory issues than for simple peptides.
[0225]The Anthrax Vaccine Expert Committee (AVEC) reviewed adverse events reported to the Vaccine Adverse Event Reporting System (VAERS) (Sever J L. Pharmacoepidemiol Drug Saf. 2002 11:189-202; Geier D A. Clin Exp Rheumatol. 2002 20:217-20). Nearly half the reports noted a local injection-site adverse effect, with more than one-third of these involving a moderate to large degree of inflammation. Six events qualified as serious adverse effects, and all were judged to be certain consequences of vaccination. Three-quarters of the reports cited a systemic adverse effect (most common: flu-like symptoms, malaise, rash, arthralgia, headache), but only six individual medically important events were judged possibly or probably due to vaccine (aggravation of spondyloarthropathy (2), anaphylactoid reaction, arthritis (2), bronchiolitis obliterans organizing pneumonia). They concluded, since some cases of local inflammation involved distal paresthesia, AVEC recommends giving subcutaneous injections of AVA over the inferior deltoid instead of the triceps to avoid compression injury to the ulnar nerve.
[0226]Ii-Key/LF(MHC Class II epitope) hybrids will induce strong Th1 immune responses that will in turn augment CTL activity, macrophage-mediated bacteria lysis, and B cell-mediated antibody production. The resulting immune responses will mediate destruction of the bacteria via enhanced macrophage activation. In addition, the hybrid will provide for augmented B cell activation, which, in the setting of concomitant or subsequent exposure to LF, will hasten and enhance the production of antibodies that block binding of LF to PA, thereby preventing internalization of anthrax toxin.
[0227]Prophylactic vaccination with the Ii-Key/LF(MHC II epitope) hybrid peptide vaccine will induce memory T-helper cells that, upon subsequent exposure to B. anthracis, will activate macrophages more potently and more rapidly, thereby resulting in efficient lysis and clearance of bacteria. Priming with the Ii-Key/LF(MHC II epitope) hybrid peptide vaccine will lead to an expanded population of specific T-helper cells that will more quickly and efficiently activate B cells for antibody production upon vaccination with LF vaccine or exposure to B. anthracis. Boosting with the Ii-Key/LF(MHC Class II epitope) hybrid peptide vaccine in patients previously vaccinated or previously exposed to the disease will create a robust and rapid anamnestic response involving efficient activation of macrophages and B cells. Prior vaccination with the Ii-Key/LF(MHC II epitope) hybrid will result in more rapid stimulation of T-helper cells and activation of B-cells providing for augmented and more rapid antibody production, which is critical in the neutralization of anthrax toxin, upon exposure to a classical anthrax vaccine or the infection itself.
[0228]In another aspect Ii-Key/anthrax MHCC lass II epitope/anthrax ARD hybrids can be used to create an effective blocking antibody eliciting vaccine. Compound peptide constructs consisting of ARDs from PA binding sites on LF, are designed with covalently linkage to the Ii-Key/antigenic epitope hybrids. In some instances the sequences of the MHC Class II epitope and an ARD overlap. These double hybrid constructs [Ii-Key/LF(MHC II epitope)/LF1-255(ARD)] trigger robust production of antibodies to LF1-255 via concomitant antigen-specific activation of T-helper cells and B-cells. The double hybrid construct focus and magnify the immune response on the most critical area, the PA63 binding site for LF. The antibodies produced following vaccination disrupt LF binding to PA63 and anthrax toxin internalization, thereby obviating the virulence of the disease. Methods of the process of developing immunization procedures with these Ii-Key/antigenic epitope hybrids for protection against anthrax and anthrax toxins include the following. 1. The most effective double hybrid(s) (in terms of inducing the most potent CD4+ T cell immunity and blocking the binding of LF1-255 to PA63 and entry of LF1-255 into cells) are tested in vivo in animal infection models to evaluate inhibition of bacteria growth and the virulence of the lethal toxin. 2. Immunization formulations (different doses with or without adjuvants), roots of immunization (s.c. or i.v.), and immunization schedules (with or without boosts) are evaluated in animal models. Toward application in a human trial, dose, dosage schedule, formulation, cytokine adjuvant, and basic local and systemic toxicities are evaluated in a murine protective model. 3. Activation of Th memory cells is tested in groups of immunized mice at 3, 6, 9 and 12 months for potency of CD4+ cell responses on a secondary challenge with the peptide, recombinant protein, or cDNA LF vaccine. 4. The most potent human HLA-DR restricted LF epitopes are determined for human clinical application. The most potent epitope for certain HLA-DR alleles are predicted using the Rammensee program. In as much as LF MHC Class II epitope aa576-591 might be presented by both HLA-DR1 and HLA-DR4, efforts to identify other pan-DR allele binding epitopes are made. The predicted Ii-Key/LF(HLA-DR epitope) constructs are tested for activity in ex vivo human PBMC stimulation and re-stimulation studies. Th1 and Th2 responses (double staining for CD4 and IFN-γ or CD4 and IL-4) are evaluated. 5. Double hybrids of the structure Ii-Key/LF(HLA-DR)/LF1-255(ARD) are synthesized using the most active Ii-Key/LF(HLA-DR epitope) and the most active antibody determinant (ARD). These are tested in animal toxicology and pharmacokinetics studies. 6. Clinical in vivo immunization and ex vivo PBMC re-stimulation studies in volunteers are performed with double hybrids to evaluate Th1 and Th2 responses. The several most promising double hybrids are evaluated in a subsequent clinical trial in which the induction of CD4+ T cell activation (double staining of PBMC for CD4 and IFN-γ or IL-4) and blocking antibodies are evaluated. ex vivo studies of the induced antibodies are performed to evaluate inhibition of the binding of LF1-255 to PA63 and LF1-255 entry into cells. The optimal hybrid(s) are further developed as an anthrax vaccine in clinical trials involving greater numbers of individuals. Appropriate efficacy endpoints and immunological surrogates are selected based on extensive discussion with appropriate regulatory agencies.
[0229]Ii-Key hybrid anthrax vaccines have significant advantages. (1) Safety. Since the Ii-Key hybrid vaccines are small peptides, as opposed to the full-length LF or PA protein, there is less risk of inducing unwanted immune responses against extraneous regions of the protein(s) which may be cross-reactive with normal host molecules, thereby resulting in autoimmune mediated toxicity. Peptide vaccine does not have reverse affect and thus can be safely used for large military and civilian populations; (2) Efficacy. To date, vaccines based on MHC Class II epitopes have not induced robust antigen specific immune responses primarily due to low binding efficiency. The Ii-Key hybrid technology enhances the charging efficiency of MHC Class II epitopes such that strong antigen-specific immune responses that are usually seen only in the context of concomitant IL-12 administration are observed. (3) Precise-targeting. Although current vaccines may induce high titers of polyclonal antibodies. However, these antibodies are not always against critical target, the LF binding site for PA. The Ii-Key double hybrid, Ii-Key/LF(MHC II epitope/LF1-255(ARD), will result in the production of antibodies specifically and precisely targeted to the LF binding sites for PA, thereby making efficient use of the resources brought to bear by the immune system. (4) Dual-action--the Ii-Key double hybrid will induce T-helper memory cells that will activate macrophages to effect cell-mediated bacterial lysis and clearing, as well as strong antibodies to the PA63 binding sites that will obviate the virulence of the anthrax toxin. Even in the setting of dense bacterial growth, the antibodies to PA binding sites on LF will protect from the virulent effects of the anthrax toxin. (5) Platform technology--once shown to be effective in the anthrax system, this approach is readily adaptable for use in other Category A (i.e., botulism, plague and smallpox), Category B, and Category C bioterrorism threats.
[0230]Ii-Key/LF(MHC II epitope) hybrids are designed to induce of LF-specific CD4+ T cell activation, which forms a major defense line to inhibit the growth of B. anthracis. Then the most potent Ii-Key/LF(MHC II epitope) hybrid are linked to putative ARDs of the PA63 binding site on LF to form double hybrids of the structure Ii-Key/LF(MHC II epitope)LF1-255(ARD). The ARDs are chosen from the published mapping of the sites on LF for binding to PA by mutation/binding assay (Lacy D B. J Biol Chem. 2002 277:3005-10). The linkage of Ii-Key/LF(MHC Class II epitope) hybrid to ARDs will offer strong CD4+ T cell help for the induction of antibodies to the covalently linked ARDs (Golvano J. Eur J Immunol. 1990 20:2363-6. These antibodies will bind to the surface of the PA binding sites on LF and block the binding of LF to PA63. The induction of high-titered antibodies against precisely targeted binding sites creates another line of defense, which abrogates the toxicity of B. anthracis LF, although the bacterial infection can be ongoing. MHC Class II-presented LF epitopes predicted with the SYFPEITHI program identifies three epitopes match perfectly the consensus sequence of the H-2Ek motif: LF(91-106; HISLEALSDKKKIK) (SEQ ID NO: 634) LF(249-264; EQEINLSLEELKDQR) (SEQ ID NO: 635); LF(305-320; DDIIHSLSQEEKELL) (SEQ ID NO: 636). The activity of all hybrids in T cell activation studies will be compared with epitopes unlinked to Ii-Key. T cell activation is measured by two-color staining (anti-CD4 plus anti-IFN-γ for Th1 and anti-CD4 plus anti-IL-4 for Th2). AKR or C3H mice (H-2Kk) are immunized (3 mice/group) with varying doses (0.8, 4, and 20 nmol) of the Ii-Key/LF(MHC II epitope) hybrids. The concentration of 20 nmol, used by Berzofsky and colleagues (Berzofsky, J. A. J Clin Invest. 1991 88: 876-84), induced optimal T cell proliferation. A much lower concentration of hybrids will induce the same or higher levels of T cell response. In the first experiment, the adjuvant emulsion consists of equal volumes of CFA containing 1 mg/ml of Mycobacterium tuberculosis and hybrid peptides dissolved in PBS. Mice are immunized s.c. on the left side at the base of the tail. The same amount of hybrid peptides in incomplete Freund's adjuvant (IFA) are injected into the right side at the base of the tail 9 days later. Hybrids are injected in saline intravenously according to the same schedule to test the requirement for CFA in the efficacy of hybrids. It should be noted that Ii-Key hybrids will interact directly with MHC Class II molecules on the cell surface of APCS, thereby bypassing classical MHC Class II epitope processing and rendering the adjuvant superfluous. Four days following the second injection, the activation of lymphocytes from spleen, popliteal, inguinal, and para-aortic nodes of immunized mice are determined by established two color staining for CD4 and either IFN-γ or IL-4 (Varga S M. J. Immunol. 2001 166:1554-61).
[0231]Ii-Key/LF(MHC II epitope)/LF1-255(ARD) double hybrids will produce antibodies which inhibit binding of LF to PA. The binding of LF to PA and subsequent entry of LF into cells are essential for the principal toxicity of B. anthracis infections. Blocking the binding of LF to PA is thus an effective way to control the virulence of B. anthracis. Lacy et al. have identified the PA binding sites on the surface of LF 1-255 by mutation/binding assays. Nine overlapping ARDs from these sites are synthesized in Ii-Key/MHC Class II antigenic epitope/ARD hybrids. Coupling to either a carrier or a MHC Class II epitope is required in order to induce antibodies against these short peptides (Golvano J. Eur J Immunol. 1990 20:2363-6). LF has been crystallized and its functional domains have been defined (Pannifer A D. Nature 2001 414:229-33; Lacy D B. J Biol. Chem. 2002 277:3005-10). By LF mutation and PA/LF binding experiments, Lacy et al. have mapped the PA63 binding sites on LF. Mutations clustered at two locations greatly abolish the binding of LF to PA63: aa182-188 and aa223-236. Because these two clusters are located on the surface of LF, at that exposed binding site (Lacy D B. J Biol Chem. 2002 277:3005-10), they are logically good targets for developing antibodies to block the binding of LF to PA63.
[0232]In another aspect this disclosure relates to augmenting the immune response to DNA vaccines for PA or LF. The Ii-Key/anthrax antigenic epitope hybrids of this disclosure can be applied as a prevaccine given in advance of a DNA vaccine for an anthrax-coded protein. Several examples of such vaccines follow.
[0233]Galloway and colleagues developed protection against anthrax lethal toxin challenge by immunization with plasmids encoding LF(10-254) or PA(175-764) or both (Price B M. Infect Immun. 2001 69:4509-15). Gold particles coated with either or both plasmids were gene-gun injected into mice three times at 2-week intervals. Antibody titers both PA and LF were five times greater than titers from mice immunized with either gene alone. All mice immunized with either or both plasmids survived an i.v. challenge with a lethal dose of PA+LF.
[0234]Gu and colleagues also studied comparable PA DNA vaccines (GU ML. Vaccine 1999 17:340-4). A 1:100 dilution of serum from mice immunized with PA DNA protected cells in vitro against cytotoxic concentrations of PA. 7 of 8 mice immunized three times with the PA DNA vaccine were protected against lethal challenge with a combination of anthrax protective antigen plus lethal factor. The augmentation of such immunizations with DNA vaccines for PA might be further augmented by a later boost with recombinant protective antigen. Such protein antigens will further enhance antibody production to PA because although Ii-Key hybrids augment the MHC-Class II restricted response to antigen expressed from a DNA vaccine, there is presumably not enough PA protein available extracellularly to bind to B cells for internalization and processing of MHC Class Ii epitopes to activate those B cells to progress to plasma cells and soluble immunoglobulin production.
[0235]The efficacy of Ii-Key/anthrax antigenic epitope hybrids in potentiating DNA and protein vaccines can be tested in guinea pigs, rabbits, and rhesus macaques against spore challenge by Bacillus anthracis isolates of diverse geographical origin (Fellows P F. Vaccine 2001 19:3241-7).
[0236]In another aspect the Ii-Key/anthrax MHC Class II epitope/anthrax ARD hybrids can be used to elicit antibodies which block the interaction of LF with PA required for the internalization of LF into cells. Examples of the creation and use of antibodies with such protective blocking effects follow.
[0237]Georgiou and colleagues found protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity (Maynard J A. Nat Biotechnol. 2002 20:597-601). The tripartite toxin produced by Bacillus anthracis is the key determinant in the etiology of anthrax. They engineered a panel of toxin-neutralizing antibodies, including single-chain variable fragments (scFvs) and scFvs fused to a human constant kappa domain (scAbs), that bind to the protective antigen subunit of the toxin with equilibrium dissociation constants (K(d)) between 63 nM and 0.25 mM. The entire antibody panel showed high serum, thermal, and denaturant stability. in vitro, post-challenge protection of macrophages from the action of the holotoxin correlated with the Kd of the scFv variants. Strong correlations among antibody construct affinity, serum half-life, and protection were also observed in a rat model of toxin challenge. High-affinity toxin-neutralizing antibodies can be of therapeutic value for alleviating the symptoms of anthrax toxin in infected individuals and for medium-term prophylaxis to infection.
[0238]In another aspect, this disclosure relates to Ii-Key/anthrax MHC Class II epitope/ARD hybrids to generate protective antibodies to a segment of PA binding LF for internalization into cells. Varughese and colleagues identified two such potential sites in solvent-exposed loops of domain 4 of PA (aa 679 to 693 and 704 to 723) by mutagenesis and testing of the purified proteins for toxicity in the presence of LF (Varughese M. Infect Immun. 1999 67:1860-5). Mutations were designed in these loops and were introduced by errors occurring during PCR. Substitutions within the large loop (aa 704 to 723) had no effect on PA activity. Comparisons among 28 mutant proteins showed that the large loop (aa 704 to 722) is not involved in receptor binding, whereas residues in and near the small loop (aa 679 to 693) are relevant to receptor interaction. Peptides through that small loop are good candidates for incorporation in Ii-Key/LF MHC Class II epitope/LF ARD hybrids.
[0239]Anthrax lethal factor can be used either to draw other proteins into a cell or for its toxic activity to inactivate MAP-kinase-kinase (Duesbery N S. Science 1998 280:734-7; Liu S. Cancer Res. 2000 60:6061-7; Liu S, J Biol Chem. 2001 276:17976-84).
[0240]The hybrids of this disclosure will enhance responses to subsequently administered anthrax toxoid vaccine adsorbed to alum (Pittman P R. Vaccine 2002 20:1412-20). The IM route of administering this is safe and has comparable peak anti-PA IgG antibody levels when two doses are administered 4 weeks apart compared to the licensed initial dose schedule of three doses administered 2 weeks apart.
[0241]The Ii-Key/antigenic epitope hybrids of this disclosure can be assayed in a rabbit model of inhalational anthrax (Pitt M L. Vaccine 2001 19:4768-73). A serological correlate of vaccine-induced immunity was identified in the rabbit model of inhalational anthrax. Animals are inoculated intramuscularly at 0 and 4 weeks with varying doses of Anthrax Vaccine Adsorbed ranging from a human dose to a 1:256 dilution in phosphate-buffered saline. At 6 and 10 weeks, both the quantitative anti-PA IgG ELISA and the toxin-neutralizing antibody assays were used to measure antibody levels to PA. Rabbits were aerosol-challenged at 10 weeks with a lethal dose of Bacillus anthracis spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. Results showed that antibody levels to PA at both 6 and 10 weeks were significant (P<0.0001) predictors of survival. In addition non-invasive nasal immunization can be used to vaccinate against anthrax (Gaur R. Vaccine 2002 20:2836-9). Mice were inoculated intranasally, subcutaneously or through the skin on days 0, 15 and 28 with purified PA. Intranasal and subcutaneous immunization with PA resulted in high IgG ELISA titers. High titers of IgA were observed only in intranasally immunized mice. In a cytotoxicity assay these sera protected J774A.1 cells from lethal toxin challenge.
[0242]Table 17.1 presents the deduced amino acid sequence of anthrax toxin lethal factor (GenBank gi|16974824; Pannifer A D. Nature 2001 414:229-233. (2001)). Table 17.2 presents predicted MHC Class II-presented epitopes of anthrax toxin lethal factor. Table 17.3 presents predicted MHC Class I-presented epitopes of anthrax toxin lethal factor. Designed Ii-Key/MHC Class II epitope hybrids for anthrax lethal factor are presented in Table 17.4. Table 17.5 presents designed Ii-Key/MHC Class II epitope/ARD hybrids for anthrax lethal factor. Table 17.6 presents the deduced amino acid sequence of anthrax protective antigen (GenBank gi:9280533; Cohen, S. Infect Immun. 2000 68:4549-4558). Table 17.7 presents predicted MHC Class II-presented epitopes of anthrax protective antigen. Table 17.8 presents predicted MHC Class I-presented epitopes of anthrax protective antigen. Designed Ii-Key/MHC Class II epitope hybrids for anthrax protective antigen are presented in Table 17.9. Table 17.10 presents designed Ii-Key/anthrax protective antigen MHC Class II epitope/anthrax protective antigen ARD hybrids.
TABLE-US-00099 TABLE 17.1 Deduced amino acid sequence of anthrax toxin lethal factor (SEQ ID NO: 637) 1 agghgdvgmh vkekeknkde nkrkdeernk tqeehlkeim khivkievkg 51 eeavkkeaae kllekvpsdv lemykaiggk iyivdgditk hislealsed 101 kkkikdiygk dallhehyvy akegyepvlv iqssedyven tekalnvyye 151 igkilsrdil skinqpyqkf ldvlntikna sdsdgqdllf tnqlkehptd 201 fsvefleqns nevqevfaka fayyiepqhr dvlqlyapea fnymdkfneq 251 einlsleelk dqrmlsryek wekikqhyqh wsdslseegr gllkklqipi 301 epkkddiihs lsqeekellk riqidssdfl steekeflkk lqidirdsls 351 eeekellnri qvdssnplse kekeflkklk ldiqpydinq rlqdtgglid 401 spsinldvrk qykrdiqnid allhqsigst lynkiylyen mninnltatl 451 gadlvdstdn tkinrgifne fkknfkysis snymivdine rpaldnerlk 501 wriqlspdtr agylengkli lqrnigleik dvqiikqsek eyiridakvv 551 pkskidtkiq eaqlninqew nkalglpkyt klitfnvhnr yasnivesay 601 lilnewknni qsdlikkvtn ylvdgngrfv ftditlpnia eqythqdeiy 651 eqvhskglyv pesrsillhg pskgvelrnd segfihefgh avddyagyll 701 dknqsdlvtn skkfidifke egsnltsygr tneaeffaea frlmhstdha 751 erlkvqknap ktfqfindqi kfiins
TABLE-US-00100 TABLE 17.2 Predicted MHC Class II-presented epitopes of anthrax toxin lethal factor. SEQ. Ii- ID. Peptide Pos. Sequence Score Allele Key NO: 17.2.1 501 WRIQLSPDT 3.1 1, 4 0 638 17.2.2 542 YIRIDAKVV 2.4 1 4 639 17.2.3 741 FRLMHSTDH 2.4 1, 3, 4 0 640 17.2.4 521 LQRNIGLEI 1.6 1, 8(519), 0 641 15, 15(518) 17.2.5 341 LQIDIRDSL 5.4 3 0 642 17.2.6 404 INLDVRKQY 4.5 3, 13(407) 0 643 17.2.7 677 LRNDSEGFI 4.3 3 7 644 17.2.8 129 LVIQSSEDY 3.6 4, 11(124) 0 645 17.2.9 698 YLLDKNQSD 3.0 4 8 646 17.2.10 477 YSISSNYMI 7.2 7 4 647 17.2.11 398 LIDSPSINL 6.7 7 5 648 17.2.12 595 IVESAYLIL 6.0 7 9 649 17.2.13 475 FKYSISSNY 5.5 7 2 650 17.2.14 241 FNYMDKFNE 4.7 8 6 651 17.2.15 375 FLKKLKLDI 4.2 8, 11 0 652 17.2.16 549 VVPKSKIDT 3.9 8 3 653 17.2.17 148 YYEIGKILS 3.4 11 0 654 17.2.18 416 IQNIDALLH 3.2 11 5 655 17.2.19 707 LVTNSKKFI 4.1 13 4 656 17.2.20 582 LITFNVHNR 3.9 13 5 657 17.2.21 527 LEIKDVQII 3.8 13 4 658 17.2.22 435 IYLYENMNI 7.5 15 7 659 17.2.23 71 LEMYKAIGG 4.7 15 3 660
Pos. is the first amino acid of the predicted MHC Class II-presented epitope of the specified sequence. Score is the score calculated by the ProPred program for the first of the given HLA-DRB*--01 alleles which were examined. The second listed allele is for exactly the same epitope or for an overlapping epitope for which the first amino acid position is given in parentheses.
TABLE-US-00101 TABLE 17.3 Predicted MHC Class I-presented epitopes of anthrax toxin lethal factor. PEPTIDE Pos. Sequence Score SEQ. ID. NO: 17.3.1 684 FIHEFGHAV 685.4 661 17.3.2 765 FINDQIKFI 342.2 662 17.3.3 147 VYYEIGKIL 336.0 663 17.3.4 277 HYQHWSDSL 300.0 664 17.3.5 113 LLHEHYVYA 285.7 665 17.3.6 331 STEEKEFLK 225.0 666 17.3.7 295 KLQIPIEPK 135.0 667 17.3.8 659 YVPESRSIL 126.0 668
Pos. is the first amino acid of the epitope of the listed sequence. The score is calculated with the SPEYETHEI program for HLA-A2.
TABLE-US-00102 TABLE 17.4 Designed Ii-Key/MHC Class II epitope hybrids for anthrax lethal factor. SEQ. ID. PEPTIDE Pos. Sequence NO: 17.4.1 501 Ac-LRMK-WRIQLSPDT-NH2 669 17.4.2 542 Ac-LRMK-YIRIDAKVV-NH2 670 17.4.3 741 Ac-LRMK-FRLMHSTDH-NH2 671 17.4.4 519 Ac-LRMK-LIQRNIGLEI-NH2 672 17.4.5 341 Ac-LRMK-LQIDIRDSL-NH2 673 17.4.6 404 Ac-LRMK-INLDVRKQYKRDI-NH2 674 17.4.7 677 Ac-LRMK-LRNDSEGFI-NH2 675 17.4.8 125 Ac-LRMK-YEPVQSSEDY-NH2 676
These hybrids incorporate some for the predicted MHC Class II epitopes of Table 17.3.
TABLE-US-00103 TABLE 17.5 Designed Ii-Key/anthrax lethal factor MHC Class II epitope/ARD hybrids. SEQ ID PEPTIDE Pos. Sequence NO: 17.5.1 166-184 Ac-LRMK- 677 II: 170 PYQKFLDVLNTIKNASDSD-NH2 17.5.2 190-213 Ac-LRMK- 678 II: 191; TNQLKEHPTDFSVEFLEQNSNEVQ- 203 NH2 17.5.3 200-224 Ac-LRMK- 679 II: 203, DFSVEFLEQNSNEVQEVFAKAFAYYI- 215 NH2 17.5.4 228-243 Ac-LRMK-QHRDVLQLYAPEAFN-NH2 680 II: 230
Pos. is the first and last amino acids of the LF sequence, which is incorporated into the hybrid. The first amino acid of the predicted MHC Class II epitopes are listed after II:. The MHC Class II alleles predicted with high scores to present individual epitopes are the following: 170: HLA-DRB*1301. 191: HLA-DRB*0401. 203: HLA-DRB*0401. 215: HLA-DRB*0101. 230: HLA-DRB*0101. 239:HLA-DRB*0801. Only the --10 alleles were scored with the ProPred predicting program. These peptides were chose from the segment of LF(182-236) containing interaction sites for binding to PA as indicated by loss of activity upon alanine substitutions at D182, D187, Y223, H229, L235 and Y236 (Lacy D B. J Biol Chem. 2002 277:3005-10). In these hybrids the intervening sequence is supplied by the natural sequence of LF, potentially contributing to the ARD structure. Upon identification of biological activity with any of these hybrids, additional hybrids would be tested with systematic deletions/extensions of the epitope-containing peptide sequence.
TABLE-US-00104 TABLE 17.6 Deduced amino acid sequence of anthrax protective antigen. (SEQ ID NO: 681) 1 mkkrkvlipl malstilvss tgnleviqae vkqenrllne sesssqgllg 51 yyfsdlnfqa pmvvtssttg dlsipssele nipsenqyfq saiwsgfikv 101 kksdeytfat sadnhvtmwv ddqevinkas nsnkirlekg rlyqikiqyq 151 renptekgld fklywtdsqn kkevissdnl qlpelkqkss nsrkkrstsa 201 gptvpdrdnd gipdsleveg ytvdvknkrt flspwisnih ekkgltkyks 251 spekwstasd pysdfekvtg ridknvspea rhplvaaypi vhvdmeniil 301 sknedqstqn tdsqtrtisk ntstsrthts evhgnaevha sffdiggsvs 351 agfsnsnsst vaidhslsla gertwaetmg lntadtarln aniryvntgt 401 apiynvlptt slvlgknqtl atikakenql sqilapnnyy psknlapial 351 naqddfsstp itmnynqfle lektkqlrld tdqvygniat ynfengrvrv 501 dtgsnwsevl pqiqettari ifngkdlnlv erriaavnps dplettkpdm 551 tlkealkiaf gfnepngnlq yqgkditefd fnfdqqtsqn iknqlaelnv 601 tniytvldki klnakmnili rdkrfhydrn niavgadesv vkeahrevin 651 ssteglllni dkdirkilsg yiveiedteg lkevindryd mlnisslrqd 701 gktfidfkky ndklplyisn pnykvnvyav tkentiinps engdtstngi 751 kkilifskkg yeig
TABLE-US-00105 TABLE 17.7 Predicted MHC Class II-presented epitopes of anthrax protective antigen. (SEQ ID NOS 682-699 respectively, in order of appearance) PEPTIDE Pos. SEQUENCE Allele Score 17.7.1 404 YNVLPTTSL B1, B7(405) 1.6 17.7.2 7 LIPLMALST B1 1.4 17.7.3 395 YVNTGTAPI B1, B3(392), B4, 1.1, 3.9, B7, B13(392) 4.9, 7.3, 2.8, 17.7.4 717 YISNPNYKV B1 1.0 17.7.5 697 LRQDGKTFI B3, B13(690) 6.3, 2.8 17.7.6 619 LIRDKRFHY B3, B8(617), 5.9, 5.8, B13(617) 4.7 17.7.7 610 IKLNAKMNI B3, B11(603), B13 5.3, 2.7, 4.1 17.7.8 625 FHYDRNNIA B4 4.9 17.7.9 298 IILSKNEDQ B4 3.9 17.7.10 174 VISSDNLQL B7, B15 6.8, 4.1 17.7.11 648 VINSSTEGL B7 6.8 17.7.12 161 FKLYWTDSQ B8 4.0 17.7.13 225 VKNKRTFLS B8, B13 3.5 17.7.14 96 FIKVKKSDE B8 2.7 17.7.15 752 ILIFSKKGY B13 4.9 17.7.16 47 LLGYYFSDL B15 4.2 17.7.17 663 IRKILSGYI B15 4.1 17.7.18 360 VAIDHSLSL B15 4.1
Pos. is the first amino acid of the predicted epitope. Allele is the HLA-DRB*--01 allele with a high score for presentation of the epitope. When a second allele is listed it predicts either exactly the same sequence or an overlaying sequence, the first amino acid residue position of which is given in parentheses. The score is the prediction score in the ProPred program for the given epitope and allele.
TABLE-US-00106 TABLE 17.8 Predicted MHC Class I epitopes of anthrax protective antigen. (SEQ ID NOS 700-709 respectively, in order of appearance) PEPTIDE Pos. Sequence 17.8.1 607 ILSGYIVEI 17.8.2 32 AIWSGFIKV 17.8.3 171 FLSPWISNI 17.8.4 328 RLNANIRYV 17.8.5 530 NIKNQLAEL 17.8.6 155 SLEVEGYTV 17.8.7 551 KLNAKMNIL 17.8.8 657 YISNPNYKV 17.8.9 225 VAAYPIVHV 17.8.10 352 LVLGKNQTL
Pos. is the first amino acid of the epitope of the listed sequence. The score is calculated with the SYFPEITHI program for HLA-A2.
TABLE-US-00107 TABLE 17.9 Designed Ii-Key/anthrax protective antigen MHC Class II epitope hybrids. PEPTIDE Pos. Sequence SEQ. ID. NO: 17.9.1 404 Ac-LRMK-NVLPTTSL-NH2 710 17.9.2 7 Ac-LRMK-LIPLMALST-NH2 711 17.9.3 395 Ac-LRMK-VNTGTAPI-NH2 712 17.9.4 717 Ac-LRMK-YISNPNYKV-NH2 713 17.9.5 697 Ac-LRMK-LRQDGKTFI-NH2 714 17.9.6 619 Ac-LRMK-LIRDKRFHY-NH2 715 17.9.7 610 Ac-LRMK-IKLNAKMNI-NH2 716 17.9.8 625 Ac-LRMK-FHYDRNNIA-NH2 717 17.9.9 298 Ac-LRMK-IILSKNEDQ-NH2 718 17.9.10 174 Ac-LRMK-VISSDNLQL-NH2 719
TABLE-US-00108 TABLE 17.10 Designed Ii-Key/anthrax protective antigen MHC Class II epitope/anthrax protective antigen ARD hybrids. SEQ. ID. PEPTIDE Pos. Sequence NO: 17.10.1 173-200 Ac-LRMK-ava- 720 II: 173 VISSDNLQLPELKQKSSNSRKKRSTSA G-NH2 17.10.2 212-232 Ac-LRMK- 721 II: 221, PDSLEVEGYTVDVKNKRTFLS-NH2 223, 225 17.10.3 203-232 Ac-LRMK- 722 II: 221, VPDRDNDGIPDSLEVEGYTVDVKNKRT 223, 225 FLS-NH2 17.10.4 664-684 Ac-LRMK-ava- 723 II: 664, IRKILSGYIVEIEDTEGLKEV-NH2 667, 672 17.10.5 685-705 Ac-LRMK- 724 II: 690, INDRYDMLNISSLRQDGKTFI-NH2 696
Pos. is the first and last amino acids of the PA sequence, which is incorporated into the hybrid. The first amino acid of the predicted MHC Class II epitopes are listed after II:. The MHC Lass II alleles predicted with high scores to present individual epitopes are the following: 173: HLA-DRB0401, 0701, 1501. 221 HLA-DRB0301. 223: HLA-DRB1101. 225: HLA-DRB0301, 801, 1101, 1301. 664: HLA-DRB0101, 0301. 690:HLA-DRB0401, 1101. 696:HLA-DRB0301. Only the **01 alleles were scored with the ProPred predicting program. In hybrids 17.10.2, 0.3, and 0.5 the intervening sequence is supplied by the natural sequence of PA, potentially contributing to the ARD structure. Upon identification of biological activity with any of these hybrids, additional hybrids would be tested with systematic deletions/extensions of the epitope-containing peptide sequence. Peptides 17.10.1 and 17.10.2 were chosen from the region PA(197-222) shown by Collier and colleagues to be sensitive to LF binding with alanine substitutions at K197, R200, P205, I207, I210 and K214 (Cunningham K. Proc Natl Acad Sci USA 2002 99:7049-53). Peptides 17.10.4 and 17.10.5 were chosen from the smaller loop of PA(679-693) shown by Leppla and colleagues to contain interaction sites for binding to PA (Varughese M. Infect Immun. 1999 67:1860-5). Upon identification of biological activity with any of these hybrids, additional hybrids would be tested with systematic deletions/extensions of the epitope-containing peptide sequence. Additional Ii-Key/PA MHC Class II epitope/ARD hybrids can be constructed with the peptides derived by phage display analyses to bind with PA-neutralizing antibodies. In these peptides the MHC Class II epitopes would be chosen from the best experimentally determined MHC class II-presented epitopes. Examples are presented in Table 17.11 for such constructs, using only a single MHC Class II-presented epitope.
TABLE-US-00109 TABLE 17.11 Designed Ii-Key/anthrax protective antigen MHC Class II epitope/anthrax protective antigen ARD hybrids. SEQ. ID. PEPTIDE Pos. Sequence NO: 17.11.1 II: 163 Ac-LRMK-YVNTGTAPI-NH2 725 17.11.2 209-230 Ac-LRMK-YVNTGTAPI-NH2 726 II: 222 17.11.3 655-675 Ac-LRMK-ava-YVNTGTAPI- 727 II: 655, 664, NH2 667 17.11.4 655-680 Ac-LRMK-ava-YVNTGTAPI- 728 II: 664, 667 NH2 17.11.5 666-680 Ac-LRMK-ava-YVNTGTAPI- 729 II: 667 NH2 17.11.6 693-706 Ac-LRMK-ava-YVNTGTAPI- 730 II: 693 NH2 17.11.7 688-706 Ac-LRMK-YVNTGTAPI-NH2 731 II: 693 17.11.8 686-706 Ac-LRMK- 732 II: 693 NGIKKILIFSKKGYEIG-NH2
Pos. is the first amino acid of the MHC Class II-presented epitope, for which only one example is given. The best epitopes determined experimentally are favored. The sequences following that epitope are the ARD sequences discovered by Collier and colleagues by selection and sequencing of phages which interact with PA binding antibodies. Some of those antibodies inhibit internalization of LF.
Example 18
Ii-Key/Variola B5R Protein Antigenic Epitope Hybrids
[0243]Ii-Key/smallpox antigenic epitope vaccines offer robust and relatively safe protection against smallpox, when used either alone or in combination with other vaccination methods. The potency and safety of certain other vaccines such as vaccinia virus are enhanced substantially, when preceded by one or more immunizations with an Ii-Key/smallpox antigenic epitope vaccine. Protection of a large population can be achieved with solely the use of the Ii-Key/smallpox antigenic epitope hybrid vaccine or preferably with such a vaccine in which the MHC Class II epitope is joined or overlapped in sequence with a MHC Class I-presented (cytotoxic T lymphocyte inducing) epitope and/or an antibody-recognized (virus neutralizing) epitope. Immunization with Ii-Key/smallpox antigenic epitope vaccines also improves clinical outlook for individuals infected with smallpox virus without prior vaccinia immunizations. The Ii-Key/antigenic epitope hybrid vaccines will enhance the protective responses of persons receiving a preventative vaccine with either vaccinia virus or a DNA for a smallpox or vaccinia viral protein. The efficacy of vaccinia virus vaccines given to individuals immediately upon exposure or potentially exposure to smallpox ("ring vaccination"), will be accelerated in terms of the speed and potency of the protective response. The biology and clinical course of smallpox infections is reviewed in order to understand the substantial benefits brought to the prevention of smallpox by the products and methods of this Disclosure.
[0244]Variola major, the smallpox virus, belongs to the family Poxyiridae, subfamily Chordopoxyirinae, and genus orthopoxvirus, which includes vaccinia (the smallpox vaccine), monkey poxvirus, and several others animal poxviruses that cross-react serologically (Breman J G. N Engl J Med. 2002 346:1300-8; Moss B. in Fields B N. Fields Virology. 1996: 2637-71; Fenner F. in Fields B N. Virology. 1996: 2673-83). The poxviruses are among the largest viruses known, containing one linear, double-stranded DNA molecule of 130 to 375 kb and replicating inn the cytoplasm.
[0245]There are five patterns of smallpox infections. Variola major (ordinary smallpox) was responsible for 90% of cases in the pre-eradication era and is associated with an overall case-fatality rate of 30% (15% to 45%) in unvaccinated patients. Flat-type or malignant smallpox and hemorrhagic smallpox typically occur in patients with a defective immune system, and case fatality rates are 97% and 96% respectively. Smallpox in children is generally similar to smallpox in adults except the case fatality rate in infants is over 40%. Variola minor is the mildest form that predominated in outbreaks in the U.S. and Great Britain, with case fatality rates <1% (Fenner F. Bull WHO. 1988 1-68, 121-208; Henderson D A. JAMA. 1999 281:2127-39).
[0246]The smallpox virus enters through the respiratory tract, passing rapidly to lymph nodes to multiply in the reticuloendothelial system over 14 days. Mucous membranes in the oropharynx become infected, as well as the capillary epithelium of the dermis leading to skin lesions. Oropharyngx and skin lesions contain abundant viral particles; virus is also present in the urine and conjunctival secretions. Cytotoxic T-cells and B-cells arise to limit the infection; neutralizing antibodies appear in the first week of infection but are delayed if infection is severe (Fenner F. in Fields B N. Virology. 1996: 2673-831996; Roberts J A. Br J Exp Pathol. 1962 43:451-61; Bedson H S. J Pathol Bacteriol. 1963 85:1-20; Buller R M. Microbiol Rev. 1991 55:80-122; Zaucha G M. Lab Invest. 2001 81:1581-600; Sarkar J K. Bull World Health Organ. 1973 48:517-22). The incubation period is 7 to 17 days (mean 10 to 12). The prodromal phase, which lasts for two to three days, is characterized by severe headache, backache, and fever, all beginning abruptly (Dixon C W. Smallpox. London, 1962). Enanthema of the tongue, mouth, and oropharynx precede the rash by a day. The rash begins as small, reddish macules, which become papules with a diameter of 2 to 3 mm. The papules become vesicles with a diameter of 2 to 5 mm. Pustules of 4 to 6 mm diameter develop four to seven days after the rash. Smallpox lesions with a peripheral distribution, generally are all at the same stage of development (in contrast to chicken pox lesions). Lesions on the palms and soles persist the longest. Death from smallpox is ascribed to toxemia, associated with immune complexes, and hypotension secondary to fluid and protein loss.
[0247]Variola is transmitted predominantly from person to person by droplet inhalation, most commonly among those with close face-to-face contact (Fenner F. Bull WHO. 1988 1-68, 121-208). Airborne and fomite (laundry, bedding) transmission occurs (Dixon C W. Smallpox. London, 1962). Patients are infectious from the time of fever onset, immediately prior to rash development. Secondary attack rates range from 37% to >70% (Rao A R. Indian J Med Res. 1968 56:1826-54; Arnt N. Am J Epidemiol. 1972 94:363-70; Heiner G G. Am J Epidemiol. 1971 94:316-26), with a primary case infecting 3.6 to 6 others (Gani R. Nature. 2001 414:748-51). In the 1970s outbreaks in Yugoslavia and Germany, there were 11 to 38 infected contacts per index case (Fenner F. Bull WHO. 1988 1-68, 121-208). Thus in populations with low herd immunity, transmission rapidly creates outbreak cases before control measures take hold. Infectivity lasts until all lesions have scabbed over and the scabs have fallen off.
[0248]Patients with smallpox are treated supportively--adequate fluid intake (which is difficult due to oropharyngeal enanthema), alleviation of pain and fever, keeping skin lesions clean to prevent bacterial superinfection. Although no antivirals are approved for smallpox by the U.S. FDA, many compounds have been screened for therapeutic activity. Cidofivir (Vistide®, approved for CMV retinitis) shows activity against orthopoxviruses, including variola (CIDRAP/IDSA. 2002).
[0249]Smallpox vaccination began in China in 1000 AD with "variolation", administration of infectious material from an infected patient to uninfected individuals. Edward Jenner discovered in the late 1700s that cowpox protected against smallpox. Vaccinia virus, genetically distinct from cowpox, has replaced cowpox as a vaccine (CIDRAP/IDSA. 2002). Protection is afforded for 5-10 years after primary vaccination; neutralizing antibodies are detected up to 10 years in 75% of individuals receiving 2 doses of vaccine, and up to 30 years in those vaccinated with 3 doses (Henderson D A. JAMA. 1999:281:2127-39). After an intensive worldwide campaign initiated in earnest in 1967, smallpox eradication was declared in 1980. With no natural reservoirs, variola has since existed only in laboratories. The WHO has sanctioned two depositories--The Center for Disease Control and Prevention (Atlanta, Ga.) and the State Research Center of Virology and Biotechnology (the Vektor Institute) in Novosibirsk, Russia. Inappropriately available variola virus could be a weapon of terrorists. Since less than 20% of 157 million individuals vaccinated before the early 1970s (when routine vaccination was discontinued in the US) are protected today and 119 million Americans have never been vaccinated, the need and problems of vaccinating against smallpox are being considered most carefully.
[0250]The Working Group on Civilian Biodefense has identified a number of widely known organisms that could cause disease and deaths in sufficient numbers to cripple a city or region. Smallpox used as a biological weapon, is perhaps the most serious threat to civilian populations due to its ease of transmission, case-fatality rate of 30% or more among unvaccinated persons, and the absence of a specific therapy. Although smallpox has long been feared as the most terrible of all infectious diseases, its potential for devastation today is much greater than at any previous time. Routine vaccination throughout the US ceased 25 years ago. In a now highly susceptible, mobile population, smallpox would spread widely and rapidly throughout this country and the world (Henderson D A JAMA. 1999 281:2127-39; Fenner F. Bull WHO. 1988 1-68, 121-208).
[0251]The U.S. vaccinia vaccine since the 1970s, Dryvax, is a lyophilized live vaccinia virus preparation manufactured by Wyeth. The vaccine is administered on a bifurcated needle containing a droplet of the reconstituted product; the skin of the upper arm is poked approximately 15 times creating a wound producing a drop of blood. To elicit a protective response, a "Jennerian pustule" must be induced. In an effort to expand current supplies in light of bioterrorism threats, recent clinical trials have tested the protective effects of Dryvax at dilutions of 1:1, 1:5, 1:10, and 1:100 (Frey S E. N Engl J Med. 2002 346:1265-75; Frey S E. N Engl J Med. 2002 346:1275-80). A major response was observed in 95% with undiluted product, 70% with 1:10 diluted vaccine, and 15% with 1:100 diluted vaccine. One month after vaccination, 34 of the 36 subjects with major reactions developed antibody responses compared to 1 of 24 patients who did not develop Jennerian pustules (Frey S E. N Engl J Med. 2002 346:1275-80). Vigorous cytotoxic T-cell and IFN-a responses occurred in 94% of subjects with major reactions and only 1 of 24 patients who did not develop Jennerian pustules.
[0252]Routine vaccination was discontinued in 1979 because the risk of complications from the vaccine outweighed the threat of endemic smallpox (Fenner F. Bull WHO. 1988 1-68, 121-208). A 10 state study indicated that there were 1254 complications per 1 million primary vaccinations including encephalitis, progressive vaccinia, eczema vaccinatum, generalized vaccinia, and erythema multiforme (Lane J M. J Infect Dis. 1970 122:303-9). A nationwide survey showed that the case fatality rate was 1 per 1 million primary vaccinations (Lane J M. N Engl J Med. 1969 281:1201-8). Certain groups of individuals are contraindicated to be vaccinated--those with conditions causing immunodeficiency (i.e., HIV infection, leukemia, lymphoma, generalized malignancy, agammaglobulinemia, organ transplant recipients, or therapy with alkylating agents, antimetabolites, radiation, or large doses of corticosteroids), persons with eczema, persons with household contacts who are immunodeficient or who have a history of eczema, and pregnant women.
[0253]Based on the observed morbidity and mortality associated with vaccinia vaccination in the US from 1967 to 1979, a mass smallpox preventative vaccination campaign in the U.S. general public aged 1 to 65 could result in as many as 4,600 serious adverse events and 285 deaths (excluding high-risk persons and their immediate contacts) (Kemper A R. Eff Clin Pract. 2002 5:84-6). Indeed, dictating that everyone receives the Dryvax vaccine would sentence as many as 400 people to death and many others to seriously debilitating side effects (Grand Rapids Press Apr. 10, 2002). Therefore, the CDC has recommended a "ring vaccination" or containment strategy. In this approach, the following individuals receive the vaccine following actual or potential release of variola virus: persons directly exposed to the release; persons with face-to-face or household contact with an infected patient or in close proximity (within 2m); personnel directly involved in the evaluation, care, or transport of infected patients; laboratory personnel involved in processing specimens; and others likely to have contact with infectious materials (CDC Interim Smallpox Response Plan CDC November 2001; Vaccinia ACIP Morb Mortal Wkly Rep. 2001 50:1-25).
[0254]Compared to mass vaccination, ring vaccination is clearly not optimal the following reasons. (1) Pre-emptive voluntary vaccination eliminates the value of smallpox as a weapon, serving as an effective deterrent. (2) Ring vaccination is effective only for the eradication of small, localized outbreaks in a population with widespread immunity. In a largely non-immune mobile population, epidemic control after multiple simultaneous exposures is a vastly different challenge. (3) Ring vaccination requires prompt identification and vaccination of infected individuals within the 3-day post exposure period when the vaccination might be effective. A person might be infective for several days before smallpox is clinically obvious, therefore, identification of cases of exposure to an infected terrorist, for example, within a four-day period is logistically impossible. (4) The CDC is assuming that each infected person will infect only 2 to 3 others, however, as many as 38 secondary infections have been observed. (5) The logistical complexity of administering millions of vaccine doses in an acute emergency is daunting and likely to induce panic and collapse of the medical and public health service as was observed in the Dark Winter simulation exercise conducted by Johns Hopkins University in June 2001 (Bicknell W J. N Engl J Med. 2002 346: 1323-25; Henderson D A. JAMA. 1999 281:2127-39; Millar J D. Public Health Policy Advisory Board. 2000; Fenner F. Bull WHO. 1988:1-68, 121-208; O'Toole T. Johns Hopkins Center for Civilian Biodefense Strategies. 2001). In contrast, pre-exposure vaccination does not pose the logistical difficulties of vaccination during an outbreak and is less expensive. In addition, pre-exposure vaccination reduces the risk of infection among immunocompromised persons (Rosenthal S R. Emerg Infect Dis. 2001 7:920-6).
[0255]Improved vaccines capable of safely and rapidly eliciting long-lasting immunity against smallpox in all persons are clearly needed. Whether used in mass or ring vaccination strategies, greater safety and efficacy relative to Dryvax is required. The Ii-Key/antigenic epitope hybrid used alone or in combination with DNA vaccines will have the following preferred characteristics relative to Dryvax: (1) significantly reduced complication rate including death and debilitating side effects, (2) more rapid induction of protective antibodies and viral-specific cytotoxic T-cells (3) simpler vaccination method, (4) greater period of protection following primary vaccination, and (5) broader target population including use in immunocompromised individuals and in pregnancy.
[0256]One preferred approach to protecting large populations is administration of one or more immunizations with an Ii-Key/smallpox antigenic epitope hybrid of this Disclosure, followed according to the ring immunization concept by vaccinia or similar viral vaccines in the population subset of exposed or potentially exposed individuals. However, in addition, when individuals who were not in the immunized ring, contract smallpox, significant protection is afforded by prior expansion and memory of CD4.sup.+ T helper cell clones, CD8.sup.+ cytotoxic T lymphocyte clones, and B cell immunoglobulin producing clones as the case might be. Such responses create a more rapid time frame for development of clinically protective responses frame to presentation of those same and other epitopes by the smallpox virus, than would be the case in individuals not immunized with the hybrids. The process of inducing responses to viral epitopes other than that in the immunizing Ii-Key/smallpox antigenic epitope hybrid, is referred to as epitope spreading.
[0257]Although vaccination is generally regarded to be the best defense against smallpox virus, the approved vaccines and some in development are not optimally safe or potent. The Ii-Key/smallpox MHC Class II epitope hybrid vaccines can be used either alone or together with other approaches, including whole virus preparations, DNA and RNA vaccines, inactivated whole virus, and virus-like particles. The Ii-Key/antigenic epitope hybrid vaccines revealed in this Disclosure can be used in conjunction with diluted whole virus preparations, e.g., Dryvax, in order to improve the major reaction rate typically observed with diluted preparations and allow for decreased rates of complications (Frey S E. N Engl J Med 2002 346:1265-75; Frey S E. N Engl J Med 2002 346:1275-80). In addition, Ii-Key/smallpox MHC Class II epitope hybrid vaccines can be used with attenuated virus strains that have been developed (Ankara MVA and Japanese strain LC16m8) in order to augment their efficacy (Rosenthal S R. Emerg Infect Dis 2001 7:920-6; Henderson D A JAMA. 1999:281:2127-39). Ii-Key/smallpox MHC Class II epitope hybrid vaccines can be used with DNA or RNA vaccines targeting gene products that are critical for viral pathogenicity and infectivity, for example, B5R and others (Phillpotts R J. Acta Virol 2000 44:151-6; Mathew E C. J Gen Virol 2001 82:1199-213).
[0258]Ii-Key/smallpox antigenic epitope hybrids offer potent and safe vaccines against smallpox. One favored example uses Ii-Key/antigenic epitope hybrids containing the Ii-Key LRMK (SEQ ID NO: 3) motif and an MHC Class II epitope of the smallpox B5R gene product gp42. Such a construct can be further enhanced with a linked or overlapping MHC Class I epitope(s) and/or antibody-determined epitope(s). By boosting the Th response >200 times to the MHC Class II epitope, Th1 cells are recruited to elicit potent CTL and humoral responses with immunological memory. Addition of a MHC Class I epitope to the hybrid affords antigenic epitope-specific enhancement of the cytotoxic T lymphocyte response. Addition of an antibody-recognized epitope to the hybrid affords antigenic epitope-specific enhancement of the antibody-determined response.
[0259]Smallpox gp42 is selected for several reasons. (1) Gene B5R encodes a 42 kD glycoprotein that is expressed throughout the course of infection and forms part of the envelope of the extracellular virus. (2) gp42 is required for the envelopment and egress of extracellular virus and virus virulence. (3) gp42-specific IgG neutralizing antibodies are correlated with protection against orthopox infection in humans (Phillpotts R J. Acta Virol 2000 44:151-6; Englestad M. Virology. 194:627-37; Mathew E C. J Gen Virol 2001 82:1199-213). In the course of routine experimentation to identify the biologically function and vaccine potential of additional proteins coded for or induced by the smallpox virus, additional candidates for the design, synthesis and use of Ii-Key/smallpox antigenic epitope hybrids will be targeted. The methods of this Disclosure can be applied without undue experimentation toward the development of additional Ii-Key/smallpox antigenic epitope hybrid vaccines. Other extracellular envelope proteins such as A33R, A34R, A36R, and A56R, can be used to produce Ii-Key/antigenic epitope hybrids.
[0260]In addition to the above vaccine methods, the Ii-Key/smallpox antigenic epitope hybrids can be used to enhance responses to DNA vaccines encoding B5R gp42. Such DNA vaccines can also be enhanced further by incorporating the Ii reverse gene construct in the same plasmid or delivery construct. Suppression of Ii protein expression allows for the presentation of endogenous gp42 epitopes. In the context of B5R DNA vaccination, targeted Ii-suppressed antigen presenting cells will present an increased repertoire of novel, perhaps cryptic, B5R epitopes.
[0261]This invention relates in part to the design of Ii-Key/Variola B5R protein antigenic epitope hybrids. The genes of the variola virus have been identified and sequenced principally by investigators in Russia (Shchelkunov S N. FEBS Lett. 1993 319:80-83; Shchelkunov S N. Virus Res. 1994 34:207-236; Shchelkunov S N. Virus Genes 1995: 9:231-245; Shchelkunov S N. Virus Res. 1996 40:169-183). The sequence of Variola virus B5R protein (g510228) is presented in Table 18.1. Predicted MHC Class II-presented epitopes of the B5R protein are presented in Table 18.2. Table 18.3 lists Ii-Key/variola B5R protein epitope hybrids containing some of the MHC Class II-presented epitopes of Table 18.2. Predicted MHC Class I-presented epitopes of variola B5R protein are presented in Table 18.4. Table 18.5 lists Ii-Key/MHC Class II-presented/MHC Class I-presented B5R hybrids.
TABLE-US-00110 TABLE 18.1 Deduced amino acid sequence of the B5R protein of the variola virus. (SEQ ID NO: 733) 1 mktisvvtll cvlpavvyst ctvptmnnak ltstetsfnd kqkvtftcds 51 gyysldpnav cetdkwkyen pckkmctvsd yvselynkpl yevnaiitli 101 ckdetkyfrc eekngntswn dtvtcpnaec qslqldhgsc qpvkekysfg 151 ehitincdvg yevigasyit ctanswnvip scqqkcdips lsnglisgst 201 fsiggvihls cksgfiltgs psstcidgkw npvlpicirs neefdpvedg 251 pddetdlskl skdvvqyeqe iesleatyhi iivaltimgv iflisvivlv 301 cscnknndqy kfhklll
TABLE-US-00111 TABLE 18.2 Predicted MHC Class II-presented epitopes of the B5R protein. SEQ Ii- ID PEPTIDE Pos. Sequence Key Score Allele NO: 18.2.1 289 VIFLISVIV 6 2.2 01 734 18.2.2 290 IFLISVIVL 7 3.8 03, 07, 15 735 18.2.3 291 FLISVIVLV 8 6.3 07 736 18.2.4 51 YYSLDPNAV 4 2.2 01 737 18.2.5 229 WNPVLPICI 13 2.1 01, 07 738 18.2.6 206 IHLSCKSGF 4 4.8 03 739 18.2.7 281 IVALTIMGV 0 4.2 03, 11, 13, 15 740 18.2.8 279 IIIVALTIM 0 3.8 03 741 18.2.9 214 FILTGSPSS 0 3.8 04 742 18.2.10 175 WNVIPSCQQ 0 3.6 04 743 18.2.11 52 YSLDPNAVC 5 3.4 04, 08, 11 744 18.2.12 277 YHIIIVALT 11 3.5 08, 11 745 18.2.13 284 LTIMGVIFL 0 2.3 08, 07, 13, 15 746 18.2.14 6 VTLLCVLPA 0 3.8 06, 01, 13 747 18.2.15 84 LYNKPLYEV 6 2.5 14 748 18.2.16 289 VIFLISVIV 6 3.6 15 749
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. When a given sequence is predicted to be presented by multiple HLA-DR alleles, the first residue position of each sequence is indicated. Score is the score reported by the ProPred program, for the relative likelihood of being presented by the first HLA-DR allele listed. The respective alleles are in each case the HLA-DRB*--01 allele. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
TABLE-US-00112 TABLE 18.3 Ii-Key/variola B5R epitope hybrids containing some of the MHC Class II-presented epitopes of Table 18.2. SEQ ID PEPTIDE Pos. Sequence NO: 18.3.1 289, 290, Ac-LRMK-ava-VIFLISVIVLV-NH2 750 291 18.3.2 16 Ac-LRMK-ava-YYSLDPNAV-NH2 751 18.3.3 229 Ac-LRMK-ava-WNPVLPICI-NH2 752 18.3.4 206 Ac-LRMK-ava-IHLSCKSGF-NH2 753 18.3.5 279, 281 Ac-LRMK-ava-IIIVALTIMGV-NH2 754 18.3.6 214 Ac-LRMK-ava-FILTGSPSS-NH2 755 18.3.7 175 Ac-LRMK-ava-WNVIPSCQQ-NH2 756 18.3.8 52 Ac-LRMK-ava-YHIIIVALT-NH2 757 18.3.9 277 Ac-LRMK-ava-YHIIIVALT-NH2 758 18.3.10 284 Ac-LRMK-ava-LTIMGVIFL-NH2 759 18.3.11 6 Ac-LRMK-ava-VTLLCVLPA-NH2 760 18.3.12 84 Ac-LRMK-ava-LYNKPLYEV-NH2 761 18.3.13 289 Ac-LRMK-ava-VIFLISVIV-NH2 762
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. In cases of closely overlapping predictions, the first residue position is given for each predicted epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 18.2.
TABLE-US-00113 TABLE 18.4 Predicted MHC Class I-presented epitopes of variola B5R protein. PEPTIDE Pos. Sequence Score SEQ ID NO: 18.4.1 292 FLISVIVLV 736 763 18.4.2 8 TLLCVLPAV 592 764 18.4.3 74 KMCTVSDYV 474 765 18.4.4 286 TIMGVIFLI 71 766 18.4.5 9 LLCVLPAVV 48 767 18.4.6 12 VLPAVVYST 29 768 18.4.7 290 VIFLISVIV 25 769 18.4.8 282 IVALTIMGV 24 770 18.4.9 77 TVSDYVSEL 18 771 18.4.10 195 LISGSTFSI 14 772
Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope predicted for HLA-A201 (Parker K C. J. Immunol. 152:163-175). Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). The MHC Class I-presented epitopes of this Table were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/).
TABLE-US-00114 TABLE 18.5 Ii-Key/MHC Class II-presented/MHC Class I- presented B5R hybrids. SEQ ID PEPTIDE Pos. Sequence NO: 18.5.1 II: 289, 290, 291 Ac-LRMK-ava- 773 I: 290, 292 VIFLISVIVLV-NH2 18.5.2 II: 286, 289, Ac-LRMK-ava- 774 290, 291 TIMGVIFLISVIVLV-NH2 I: 290, 292 18.5.3 II: 277, 279, 284 Ac-LRMK-ava- 775 I: 286 YHIIIVALTIMGVIFLI-NH2 18.5.4 II: 6 Ac-LRMK-ava- 776 I: 8, 9 VTLLCVLPAVV-NH2
Pos. is the residue position in the primary sequence of the first amino acid in either the MHC class II-presented antigenic epitope (II:) or the MHC class I-presented antigenic epitope (I:). Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II-presented epitope of Table 18.2 and a MHC Class I-presented epitope of Table 18.5.
Example 19
Ii-Key/Ebola Virus Antigenic Epitope Hybrids
[0262]Being among the most virulent infectious agents known, the Filoviruses, which include the Marburg and Ebola viruses, are classified at biosafety level 4 due to the extreme pathogenicity of certain strains and the absence of a protective vaccine or effective antiviral drug (Wilson J A. Cell Mol Life Sci. 2001 58:1826-41). Ebola virus causes a hemorrhagic fever, a severe, mostly fatal disease in humans and nonhuman primates, recognized in sporadic clusters since 1976. The natural reservoir for Ebola virus is an animal native to Africa (Peters C J. J Infect Dis. 1999 179(Suppl 1): ix-xvi). The strain Ebola-Reston was isolated in the U.S. from imported cynomologous monkeys. Public concern over Ebola virus in non-African countries is derived from potential for spread of the viruses by international commerce, jet travel, and bioterrorism.
[0263]Clusters of Ebola virus infections in humans appear to depend upon the first patient contacting an infected animal. After an index case-patient is infected, transmission occurs among humans by direct contact with (1) blood and/or secretions of an infected person and (2) objects, such as needles and syringes, that have been contaminated with infected secretions. All Ebola viruses can be transmitted in aerosols under research conditions.
[0264]Within a few days of becoming infected with Ebola virus, most patients have high fever, headache, myalgia, abdominal pain, fatigue and diarrhea. Some patients have sore throat, hiccups, rash, red eyes, and bloody emesis and diarrhea. Within a week of becoming infected with Ebola virus, most patients have chest pain, shock, and death, while some experience blindness and bleeding (Gear H S. Reviews of Infectious Diseases. 1989 11(suppl 4):5777-5782). Why some patients recover from Ebola hemorrhagic fever is not understood, although those who do develop a significant immune response to the virus.
[0265]Treatment of patients with Ebola hemorrhagic fever is supportive, consisting primarily in balancing the patient's fluids and electrolytes, maintaining oxygenation and blood pressure, and treating accompanying infections (CDC. Management of patients with suspected viral hemorrhagic fever. Morbidity and Mortality Weekly Report. 1988 37(suppl 3):1-16).
[0266]Ebola virus has caused a series of devastating hemorrhagic fever outbreaks, the first being reported in 1976 in Yambuku, Zaire where 318 people contracted Ebola hemorrhagic fever, with 88% dying. Disease spread by close personal contact and contaminated needles and syringes in hospitals and clinics. Also in 1976, in the Sudan (Nzara and Maridi), 284 people contracted Ebola hemorrhagic fever, with 53% dying and the disease being spread mainly through close personal contacts in hospitals (Bowen E T W. Lancet 1977 1:571-3). In 1979, there was a recurrent outbreak in the Sudan, with 34 patients and 65% dying (Baron R C. Bull WHO 1983 62:997-1003). In 1994, 44 people in Gabon (Minkebe, Makokou, and gold-mining camps deep within the rain forest) developed Ebola hemorrhagic fever, with 63% dying. This outbreak was thought initially to be yellow fever, however in 1995 it was identified to be Ebola hemorrhagic fever (Georges A J. J Infect Dis. 1999 179 Suppl 1:S65-75). In 1995, 315 people in Kikwit, Democratic Republic of the Congo (formerly Zaire) contracted Ebola hemorrhagic fever, with 81% dying (Le Geuenno B. Lancet. 1995 345:1271-4). This outbreak was traced to an index case-patient who worked in a forest adjoining the city; the outbreak spread through families and hospitals. In 1996 in Mayibout, Gabon, 37 people developed Ebola hemorrhagic fever, with 57% dying. A dead, infected chimpanzee, eaten by 19 people in the forest, initiated this outbreak. In the same year in Boue, Gabon, 60 patients were infected with Ebola-Zaire, with 75% dying. The index case-patient was a hunter who lived in a forest camp; a dead, infected chimpanzee was found nearby. Finally, in 2000 and 2001 in Uganda (Gulu, Masindi, and Mbarara) 425 people contracted Ebola hemorrhagic fever, with 53% dying. The three most important risk factors associated with infection were: attending funerals of Ebola hemorrhagic fever case-patients, contacting case-patients in one's family, and providing medical care to Ebola case-patients without using adequate personal protective measures and practices (CDC: SPB: Disease Information Fact Sheets: Ebola: Case Table 2001).
[0267]Because the natural reservoir for Ebola virus is undetermined and human-to-human spread is documented, vaccines appear to be the best method to limit infectious spread (Nabel G L. Trans Am Clin Climatol Assoc. 2001 112:79-84). Antibodies isolated from serums of patients recovered from the 1995 Ebola infection Kikwit, Democratic Republic of the Congo, using recombinant phage display adsorption techniques, neutralized Ebola infectivity (Maruyama T. J Virol. 1999 73:6024-30). This finding coupled with the fact that dying patients do not mount an immunologically potent response offers hope that preventative vaccines will be effective. While no such vaccines are available, several vaccine approaches are under development including DNA and RNA replicon vaccines encoding Ebola viral proteins NP (major nucleocapsid protein), VP35 (phosphoprotein), VP40 (membrane-associated matrix protein), GP (transmembrane glycoprotein), sGP (secreted glycoprotein), VP30 (ribonucleoprotein associated--minor), and VP24 (membrane-associated protein--minor) (WO 99/32147; WO 00/00617; Wilson J A. Virology. 2001 286:384-90; Pushko P. Vaccine. 2000 19:142-53; and Vanderzanden L. Virology. 1998 246:134-44).
[0268]The NIAID plans to initiate clinical trials with an adenoviral vaccine encoding genes for Ebola glycoprotein and nucleoprotein within 2 years. This vaccine induces protective immunity in non-human primate studies (Sullivan N J, Nature 2000 408:605-9; Cheary M, Dutch Firm to Develop Ebola Vaccine with US, Reuters May 16, 2002). Another vaccine is being developed with Ebola-like particles which are nonreplicating due to the absence of Ebola genetic materials, but possessing proteins contained on the inner and outer membranes (UASAMRIID, Bavari S, J Exp Med. 2002 195:1-11). A variety of vaccine strategies that protected mice and guinea pigs from lethal challenges with Ebola virus have been tested in non-human primates including: RNA replicon particles derived from attenuated strain of VEE expressing Ebola glycoprotein and nucleoprotein, recombinant Vaccinia virus expressing Ebola glycoprotein, liposomes containing lipid A and inactivated Ebola virus, and a concentrated inactivated whole Ebola virion preparation (Geisbert T W. Emerg Infect Dis. 2002 8:503-7; Pushko P. J Virol. 2001 75:11677-85; and Pushko P. Vaccine. 2000 19:142-53). Unfortunately, none of these approaches were successful in protecting non-human primates from lethal Ebola virus challenge.
[0269]Vaccinating nice with Venezuelan equine encephalitis (VEE) virus replicons encoding Ebola virus nucleoprotein induced both antibodies and MHC Class I-restricted cytotoxic T-cells to an 11 amino-acid, Ebola virus NP(43-53). Passive transfer of polyclonal antibodies did not protect mice from a lethal challenge with Ebola virus; however, adoptive transfer of Ebola virus NP-specific CTLs did protect mice from an Ebola virus lethal challenge (Wilson J A. J Virol. 2001 75:2660-4). Protective recombinant antibodies have been identified to 5 unique epitopes of Ebola glycoprotein, with one of the epitopes being conserved among all strains known to be pathogenic for humans (Wilson J A. Science. 2000 287:1664-6). Some of those monoclonal antibodies were also therapeutically effective upon administration to mice 2 days following a lethal challenge with Ebola virus. These data support view that both antibody and cell-mediated responses are important for protection against Ebola virus and therefore vaccine strategies designed to promote both antibody and CTL responses are preferred.
[0270]Although vaccines are generally regarded to be the best defense against Ebola virus, vaccines in development have not been demonstrated to be optimally protective. In the case of DNA vaccines, whether presented in plasmids, in viral particles, or in another formulation, some of these developmental issues include: 1) delivery vector of formulation (cDNA as naked DNA, or in plasmid or bacterial vectors, or with lipid or other transfecting carrier, or on gold particles or in PLG particles), 2) route of administration (skin, mucosal (GI or respiratory tracts), or muscle) 3) choice or one or multiple EBOLA genes and promoters for those genes, 4) genetic or protein adjuvants for cytokines or the products of this Disclosure, 5) dose, dosage schedule and other pharmacokinetic and pharmacodynamic considerations.
[0271]This example presents the design of a potent and relatively safe vaccine against Ebola virus VP24. The deduced amino acid sequence of Ebola VP24 is from GenBank g16751326 (Leroy E M. J Gen Virol 2002 83: 67-73). The strain of this protein was the one present in deceased, surviving and asymptomatically infected individuals during the 1996 outbreak in Gabon. Sequences of GP, NP, VP24 and VP40 genes were obtained with comparative studies and phylogenetic characterization.
[0272]Although experimentally determined MHC Class II epitopes are a more expeditious route to the construction of Ii-Key/antigenic epitope hybrids, such can be made with epitopes predicted with algorithms. Such epitopes predicted to be presented by multiple HLA-DR alleles are presented in Table 2. Ii-Key/Ebola MHC Class II antigenic epitope hybrids containing the Ii-Key LRMK (SEQ ID NO: 3) motif and single or significantly overlapping MHC Class II epitopes of VP24 are presented in Table 3. Such hybrids can be constructed with the fusion of MHC Class I-presented epitopes. Again in the absence of experimentally determined MHC Class I-presented epitopes, algorithm-predicted epitopes have been identified (Table 4). Such epitopes can be fused into Ii-Key/MHC Class II antigenic epitope hybrids, preferably when the highest degree in overlap of the MHC Class II and MHC Class I sequences are obtained. Examples of such products are presented in Table 5. When experimentally determined, antibody-recognized determinants have been identified experimentally or by prediction, additional hybrids composed of Ii-Key/MHC Class II-presented antigenic epitopes and such antibody-recognized epitopes can be designed by the methods presented herein without undue experimentation. Furthermore, the methods applied to the design and testing of Ii-Key/antigenic epitope hybrids composed of epitopes form VP24 can also be applied to similar vaccine hybrids with epitopes form other Ebola virus proteins, such as GP, NP, sGP, VP24, VP30, VP35 and VP40. The experimental validation of these hybrids can be accomplished in vaccination studies of mice by routine methods (Wilson J A. Virology. 2001 286:384-90). Among additional objective in such murine studies is the testing of the concept that presentation of a MHC Class II-presented epitope in and Ii-Key/antigenic epitope hybrid will lead to presentation by a low responder allele, functionally converting the presentation to a promiscuous epitope, as discussed in eh Background of the Invention. In the study of Wilson J A and colleagues, although immunization with VP24 was capable of stimulating a potent immune response in a BALB/c model, VP24 induced no protective effects in the C57BL/6 strain (Wilson J A. J Virol. 2001 75:2660-4). Thus, immunization of both BALB/c and C57BL/6 strains of mice with a MHC Class II-presented VP24 epitope will yield comparable immune responses as measure by antibody titers to the epitope in ELISAs, by induction of CD4+/IFNγ+ cells in the two-color FACS analysis, and by induction of CD4+/IFNγ+ cells in ELISPOT assays. Furthermore C57BL/6 mice will be protected against a lethal challenge against VEE.
[0273]The sequence of Ebola virus membrane associated protein VP24 (GenBank #g16751326; Leroy E M. J Gen Virol 2002 83: 67-73) is presented in Table 1. Predicted MHC Class II-presented epitopes are presented in Table 2. Ii-Key/Ebola virus VP24 MHC class II epitope hybrids are presented in Table 3. Predicted Ebola virus VP24 MHC Class I-presented epitopes are presented in Table 4. Ii-Key/Ebola VP 24 MHC Class II-predicted epitope/Ebola VP 24 MHC Class I-predicted epitope hybrids are presented in Table 5.
TABLE-US-00115 TABLE 19.1 Sequence of Ebola virus membrane associated protein VP24 (SEQ ID NO: 777) 1 makatgrynl ispkkdlekg vvlsdlcnfl vsqtiqgwkv ywagiefdvt 51 hkgmallhrl ktndfapaws mtrnlfphlf qnpnstiesp lwalrvilaa 101 giqdqlidqs lieplagalg lisdwllttn tnhfnmrtqr vkeqlslkml 151 slirsnilkf inkldalhvv nyngllssie igtqnhtiii trtnmgflve 201 lqepdksamn rkkpgpakfs llhestlkaf tqgsstrmqs lilefnssla i
TABLE-US-00116 TABLE 19.2 Predicted MHC Class II-presented epitopes. SEQ ID PEPTIDE POS. Sequence Score DR Ii-Key NO: 19.2.1 93 LRVILAAGI 2.90 0101 11 778 19.2.2 20 VVLSDLCNF 4.90 0301 3 779 19.2.3 151 LIRSNILKF 4.30 0301 5 780 19.2.4 146 LKNLSLIRS 4.20 0301 7 781 19.2.5 157 LKFINKLDA 3.90 0301 5 782 19.2.6 135 MTRQRVKEQ 3.60 0306 0 783 19.2.7 169 VNYNGLLSS 3.40 0306 5 784 19.2.8 220 LLHESTLKA 4.30 0401 0 785 19.2.9 124 WLLTTNTNH 3.98 0401 0 786 19.2.10 187 IIITRTNMG 3.80 0401 0 787 19.2.11 28 FLVSQTIQG 3.28 0401 4 788 19.2.12 34 IQGWKVYWA 4.80 0402 11 789
The epitopes of this Table were chosen by the following procedure. The sequence of EBOLA VP24 (GenBank g16751326) was subjected to HLA-DR epitope screening with the ProPred program. The 4 highest scoring epitopes of each allele was identified. Among that set, the first 14 unique epitopes were reported here, with the HLA-DR allele of their first occurrence. Many epitopes that are reported here, were in fact scored by the sequence algorithms of several alleles. Pos. is the amino acid residue position of the first amino acid of the epitope. Score is the score calculated by the ProPred program. Ii-Key is the number of amino acid residues intervening between the first amino acid of the epitope and N-terminally, a 5-amino acid-motif containing at least two amino acids of the group LIVFM (SEQ ID NO: 790) and at least one amino acid of the group HKR.
TABLE-US-00117 TABLE 19.3 Ii-Key/Ebola virus VP24 MHC class II epitope hybrids. SEQ ID PEPTIDE POS. Sequence NO: 19.3.1 20 Ac-LRMK-VVLSDLCNF-NH2 791 19.3.2 28 Ac-LRMK-FLVSQTIQG-NH2 792 19.3.3 34 Ac-LRMK-IQGWKVYWA-NH2 793 19.3.4 28; 34 Ac-LRMK-FLVSQTIQGWKVYWA-NH2 794 19.3.5 146 Ac-LRMK-LKMLSLIRS-NH2 795 19.3.6 151 Ac-LRMK-LIRSNILKF-NH2 796 19.3.7 157 Ac-LRMK-LKFINKLDA-NH2 797 19.3.8 146; 151 Ac-LRMK-LKMLSLIRSNILKF-NH2 798 19.3.9 169 Ac-LRMK-VNYNGLLSS-NH2 799 19.3.10 220 Ac-LRNK-LLHESTLKA-NH2 800 19.3.11 Ac-LRMK-NH2 801
TABLE-US-00118 TABLE 19.4 Predicted Ebola virus VP24 MHC Class I- presented epitopes. PEPTIDE POS. Sequence Score SEQ ID NO: 19.4.1 22 VLSDLCNFL 819 802 19.4.2 241 LILEFNSSL 288 803 19.4.3 156 NILKFINKL 95 804 19.4.4 118 ALGLISDWL 58 805 19.4.5 32 SQTIQGWKV 53 806 19.4.6 144 QLSLKMLSL 49 807 19.4.7 110 SLIEPLAGA 47 808 19.4.8 221 LLHESTLKA 35 809 19.4.9 9 NLISPKKDL 21 810 19.4.10 149 MLSLIRSNI 18 811 19.4.11 121 LISDWLLTT 16 812 19.4.12 120 GLISDWLLT 13 813 These HLA*A201 epitopes were scored with a computer-assisted algorithm (Parker K C. J. Immunol. 152: 163-175).
TABLE-US-00119 TABLE 19.5. Ii-Key/Ebola VP 24 MHC Class II-predicted epitope/Ebola VP 24 MHC Class I-predicted epitope hybrids. SEQ ID PEPTIDE POS. Sequence NO: 19.5.1 II: 20; I: 22 Ac-LRMK-VVLSDLCNFL-NH2 814 19.5.2 II: 28; I: 22 Ac-LRMK- 815 VLSDLCNFLVSQTIQG-NH2 19.5.3 II: 124; Ac-LRMK-GLISDWLLTTNTNH- 816 I: 120, 121 NH2 19.5.4 II: 146; Ac-LRMK-QLSLKMLSIRS-NH2 817 I: 144 19.5.5 II: 146; Ac-LRMK-QLSLKMLSLIRSNI- 818 I: 146, 149 NH2 19.5.6 II: 157; Ac-LRMK-NILKFINKLDA-NH2 819 I: 156 19.5.7 II: 151, 157; Ac-LRMK- 820 I: 149, 156 LIRSNILKFINKLDA-NH2 _19.5.8 II: 220; Ac-LRMK-LLHESTLKA-NH2 821 I: 221
Example 20
Ii-Key/Myelin Basic Protein MHC Class II-Presented Epitope Hybrids
[0274]In another aspect, induction of suppressor T-immunoregulatory cells specific for autoantigens, such as myelin basic protein in multiple sclerosis and collagen in arthritis, is a well-investigated and promising strategy for the control of these human autoimmune diseases. Administering peptide from myelin basic protein or collagen by oral or respiratory routes decreases antibodies to these proteins, suppresses cellular immune responses, and delays or inhibits development of experimental allergic encephalitis or collagen arthritis in animal models. In addition, certain hMBP peptides, which bind to and neutralize anti-MBP antibodies, have been tested in the clinic. MBP75-85 peptide administered intrathecally neutralized anti-myelin basic protein antibodies; intravenous administration of this peptide resulted in decreased titers of free and bound anti-myelin basic protein levels through an active immunotolerance-inducing mechanism. Various peptides ranging from 10 amino acids to 25 amino acids within the MBP sequence of 61 to 106 demonstrated this activity. Such peptides and methods of their use, which can be adapted for novel therapies with Ii-Key/antigenic epitope hybrids, have been described in U.S. Pat. No. 5,858,364: H L Weiner and D A Hafler, Jan. 12, 1999--Pharmaceutical dosage form for treatment of multiple sclerosis; and U.S. Pat. No. 5,571,499: D A Hafler and H L Weiner, Nov. 5, 1996--Treatment of autoimmune disease by aerosol administration of autoantigens and U.S. Pat. No. 6,258,781: KG Warren and I Catz, Jul. 10, 2001--Peptide specificity of anti-myelin basic protein and the administration of myelin basic protein peptides to multiple sclerosis patients, the disclosures of which are incorporated herein by reference. These results have been considered in detail below with respect to the incorporation of such epitopes in to Ii-Key/antigenic epitope hybrids to increase the potency, safety, memory and Th subset preference of such therapeutic effects.
[0275]Multiple sclerosis (MS), a demyelinating apparently autoimmune disease of the central nervous system associated with inflammation and gliosis, demonstrates T lymphocytes and autoantibodies directed to myelin proteins. Immunosuppressive therapies of multiple sclerosis can be developed with peptide epitopes from several myelin proteins. Such epitopes incorporated into Ii-Key/antigenic epitope hybrids can be tested in experimental allergic encephalitis, the animal model of multiple sclerosis. These proteins include myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and myelin-associated oligodendrocyte basic protein (MOBP) (Zamvill S S. Nature. 1986 324:258-60; Kono D H. J Exp Med. 1988 168:213-27; Madsen L S. Nat Genet. 1999 23:343-7; Tuohy V K. J Immunol. 1989 142:1523-7; Greer J M. J Immunol. 1992 149:783-8; Mendel I. Eur J Immunol. 1995 25:1951-9). The MHC Class II-presented epitopes of particular therapeutic interest are summarized here and then the experimental data supporting their use in Ii-Key/antigenic epitope hybrids are reviewed in detail in part to consider methods for their use in both preclinical animal models and in the development and use of clinical therapies based on such studies. MBP85-99 is immunodominant in humans, and several epitopes in this region induce EAE in mice (MBP87-98, MBP91-104, and MBP84-102). PLP139-151 and PLP178-191 are encephalitogenic epitopes in mice; when whole protein is used to immunize mice, lymph node cells respond to both of these epitopes indicating they are co-dominant. The encephalitogenic potential of several predicted T-cell epitopes from MOG (1-21, 35-55, 67-87, 104-117, and 202-218) were tested in mice; only MOG35-55 induced specific T-cell responses and EAE. This epitope stimulates specific T cell responses to MOG40-55 and T cell lines reactive to MOG40-55 were encephalitogenic upon transfer to syngeneic mice. MOBP37-60 is encephalitogenic in mice. Peripheral blood lymphocytes from a patient with MS mount a proliferative response to MOBP, especially MOBP21-39. The use of a DNA plasmid encoding multiple encephalitogenic epitopes derived from MBP (7-50, 83-106, and 142-168), MOG (1-25, 32-58, and 63-97), and PLP (30-60, 84-116, and 139-155) was shown to protect mice from developing EAE induced by PLP139-151.
[0276]Ii-Key/antigenic epitope hybrids comprising MHC Class II-presented epitopes derived from autoantigenic peptides from MBP, PLP, MOG, and MOBP, as described above, will have many preferred characteristics as immunopharmacological therapeutics. The useful effects of such Ii-Key/antigenic epitope hybrids in the treatment of MS, whether used as peptides or DNA vaccines include the following: (1) more rapid and potent immunosuppressive responses, (2) longer-duration of immunosuppressive responses and memory for later challenges, (3) decreased incidence of neo-reactivities as a result of intra- or intermolecular spread of autoimmunity, (4) greater breadth of response as a result of more potent presentation of epitopes on otherwise low-responding alleles, and (5) greater protection against the development, or slowing or reversal of, clinical manifestations of disease.
[0277]Warren, Catz and colleagues have demonstrated that human myelin basic protein (hMBP) peptide-based tolerance induction might be an effective antigen-specific immunotherapy for MS (Warren K G. J Neurol Sci. 1995 133:85-94; Warren K G. J Neurol Sci. 1997 148:67-78; Warren K G. J Neurol Sci. 1997 152:31-8). Tolerance to myelin basic protein (MBP) was examined in a Phase I clinical trial in MS patients with chronic progressive disease using hMPB peptide P85VVHFFKNIVTP96 (SEQ ID NO: 822) that is immunodominant for MBP-specific T cells and B cells. Tolerance induction was monitored by titers of MBP-specific autoantibodies in the CSF. Intravenous but not intrathecal or subcutaneous injection induced tolerance to MBP. Four kinetic patterns of response were observed in 41 patients (Warren K G. Mult Scler. 2000 6:300-11): Group A (15 patients) illustrated prolonged anti-BMP suppression into the normal range; Group B (10 patients) illustrated significant anti-MBP suppression into the normal range for shorter durations; Group C (eight patients) showed significant CSF anti-MBP suppression after the initial injection but lost the ability to suppress the autoantibody titer following subsequent injections; and Group D (eight patients) failed to show significant CSF anti-MBP suppression. In the control group, anti-MBP antibodies remained persistently elevated over the 2-year period. Tolerance duration depended on MHC Class II haplotypes of patients; tolerance was long-lived in all patients with disease-associated HLA-DR2. No neurological or systemic side effects were observed, regardless of the route of peptide administration.
[0278]Lees and colleagues identified several encephalitogenic determinants of myelin proteolipid protein active in SJL mice (Tuohy V K. J Immunol. 1989 142:1523-7; Greer J M. J Immunol. 1992 149:783-8). Immunization with PLP, the major protein constituent of central nervous system myelin, induces an acute form of EAE SJL/J (H-2s) mice. Immunization with PL139-154 (HCLGKWLGHPDKFVGI) (SEQ ID NO: 823) induced severe clinical and histological EAE in 3 of 20 mice. In addition, PLP(178-191) also induced EAE in these mice. Two CD4+, peptide-specific, I-A(s)-restricted T cell lines, selected by stimulation of lymph node cells with either PLP 178-191 or 139-151, were each encephalitogenic in naive syngeneic mice.
[0279]Ben-Nun and colleagues tested several peptides from pMOG, finding that a myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice (Mendel I. Eur J Immunol. 1995 25:1951-9; Kaye J F. J Neuroimmunol. 2000 102:189-98). This group also tested the hypothesis that multiple potentially pathogenic antimyelin T cell reactivities could be inhibited by tolerogenic administration of an artificial "multiantigen/multiepitope" protein (Zhong M C. J Clin Invest. 2002 110:81-90). A synthetic gene was constructed to encode selected disease-relevant epitopes of myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). Systemic administration of hmTAP not only suppressed and treated experimental autoimmune encephalomyelitis (EAE) initiated by autoreactivity to a PLP epitope, but also abrogated complex EAE transferred by multispecific line T cells reactive against encephalitogenic epitopes of MBP, PLP, and MOG. In addition Oldstone and colleagues identified the MOBP37-60 epitope, which induced experimental allergic encephalomyelitis in mice with a severe clinical course (Holz A. J Immunol. 2000 164:1103-9). Also PBL from patients with relapsing/remitting multiple sclerosis mount a proliferative response to human MOBP, especially at amino acids 21-39.
[0280]Anti-myelin antibodies can be found in some patients without MS (Warren K G. Eur Neurol. 1999 42:95-104). Wucherpfenning, Catz, Warren and colleagues affinity-purified MBP autoantibodies from central nervous system lesions of 11 postmortem cases (Wucherpfennig K W. J Clin Invest. 1997 100:1114-22). The MBP(83-97) peptide was immunodominant in all cases since it inhibited autoantibody binding to MBP>95%. Residues contributing to autoantibody binding were located in a 10-amino acid segment (V86-T95) that also contained the MHC/T cell receptor contact residues of the T cell epitope. In the epitope center, the same residues were important for antibody binding and T cell recognition.
[0281]Ii-Key Hybrids comprising MBP82-98 epitopes will increase the duration of anti-MBP suppressive responses, increase the proportion of patients developing sustained anti-MBP suppressive responses, increase the magnitude of the anti-MBP suppressive response, and decrease the number and frequency of doses needed to induce clinically effective anti-MBP suppressive responses. Because epitope charging with Ii-Key Hybrids is much greater than epitope alone, Ii-Key/MBP (85-96) Hybrids will facilitate binding to a greater repertoire of HLA DR alleles, thereby increasing the proportion of patients responding to treatment. Ii-Key/MBP (MBP85-96) Hybrids will also trigger a cell-mediated suppressive immune response to the MBP85-96 epitope. Ii-Key Hybrids comprising this and/or other MHC Class II epitopes either alone, or in combination with MHC Class I or B-cell antibody recognized determinants (arranged linearly in tandem to--or imbedded within--the MHC Class II epitope) will also induce enhanced immunosuppressive responses the MBP that provide for clinically significant therapeutics for patients with multiple sclerosis.
[0282]Selected preclinical studies reveal mechanisms and specific structural (chemical) data that are useful in determining optimal epitope structure and methods of use of Ii-Key/antigenic epitope hybrids in the treatment of MS. Krogsgaard M, Wucherpfennig K W, Fugger L and colleagues used phage display technology to select HLA-DR2-peptide-specific antibodies from HLA-DR2-transgenic mice immunized with HLA-DR2 molecules complexed with MBP 85-99 (Krogsgaard M. J Exp Med. 2000 191:1395-412). The MK16-selected antibodies recognized only complexes of DR2 and MBP and recognized intra- and extracellular HLA-DR2-MBP peptide complexes when antigen-presenting cells (APCs) had been pulsed with recombinant MBP. MK16 antibodies inhibited interleukin 2 secretion by two transfectants that expressed human MBP-specific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 85-99 and the COOH-terminal part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2-MBP peptide complex was presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases with other haplotypes. These results demonstrated that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and indicate that microglia/macrophages rather than astrocytes are the predominant APCs in these lesions.
[0283]Fugger and colleagues expressed in transgenic mice three human components involved in T-cell recognition of an MS-relevant autoantigen presented by the HLA-DR2 molecule: DRA*0101/DRB1*1501 (HLA-DR2), an MHC class II candidate MS susceptibility genes found in individuals of European descent; a T-cell receptor (TCR) from an MS-patient-derived T-cell clone specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 4102 peptide; and the human CD4 co-receptor (Madsen L S. Nat Genet. 1999 23:258-9). The amino acid sequence of the MBP 84-102 peptide is the same in both human and mouse MBP. Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination that led to clinical manifestations and disease courses resembling those seen in MS. Spontaneous disease was observed in 4% of mice. When DR2 and TCR double-transgenic mice were backcrossed twice to Rag2 (for recombination-activating gene 2)-deficient mice, the incidence of spontaneous disease increased, demonstrating that T cells specific for the HLA-DR2 bound MBP peptide are sufficient and necessary for development of disease. This study provided evidence that HLA-DR2 can mediate both induced and spontaneous disease resembling MS by presenting an MBP self-peptide to T cells.
[0284]Susceptibility to multiple sclerosis is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 (DRB1*1501) haplotype. Wiley and colleagues determined the structure of HLA-DR2 was determined with a bound peptide from human myelin basic protein (MBP) that was immunodominant for human MBP-specific T cells (Smith K J. J Exp Med. 1998 188:1511-20; Gauthier L. Proc Natl Acad Sci USA. 1998 95:11828-33). Residues of MBP peptide that are important for T cell receptor recognition are prominent, solvent exposed residues in the crystal structure. A distinguishing feature of the HLA-DR2 peptide binding site is a large, primarily hydrophobic P4 pocket that accommodates a phenylalanine of the MBP peptide. The necessary space for this aromatic side chain is created by an alanine at the polymorphic DRbeta 71 position. These features make the P4 pocket of HLA-DR2 distinct from DR molecules associated with other autoimmune diseases.
[0285]The binding site orientation of Ii-Key/antigenic epitope hybrids can be proposed from analysis of the binding of TCR with hMBP/DR2 complexes. The structural basis for the specificity of ternary complex formation by the TCR and MHC/peptide complexes was examined for myelin basic protein (MBP)-specific T-cell clones restricted by different DR2 subtypes (Wucherpfennig K W. Proc Natl Acad Sci USA. 1995 92:8896-900). Conserved features of this system allowed a model for positioning of the TCR on DR2/peptide complexes to be developed: (i) The DR2 subtypes that presented the immunodominant MBP peptide differed only at a few polymorphic positions of the DR beta chain. (ii) TCR recognition of a polymorphic residue on the helical portion of the DR beta chain (position DR beta 67) was important in determining the MHC restriction. (iii) The TCR variable region (V) alpha 3.1 gene segment was used by all of the T-cell clones. TCR V beta usage was more diverse but correlated with the MHC restriction, i.e., with the polymorphic DR beta chains. (iv) Two clones with conserved TCR alpha chains but different TCR beta chains had a different MHC restriction but similar peptide specificity. The difference in MHC restriction between these T-cell clones appeared due to recognition of a cluster of polymorphic DR beta-chain residues (DR beta 67-71). MBP(85-99)-specific TCRs, therefore, appeared to be positioned on the DR2/peptide complex such that the TCR beta chain contacted the polymorphic DR beta-chain helix while the conserved TCR alpha chain contacted the nonpolymorphic DR alpha chain.
[0286]Table 20.1 presents the deduced amino acid sequence of human myelin basic protein (GenBank gi:17378805). Table 20.2 presents MHC Class II-presented epitopes of human myelin basic protein predicted with the SYFPEITHI program. Table 20.3 presents sequences identified experimentally to contain hMBP MHC Class II-presented epitopes (Pette M. Proc Natl Acad Sci USA. 1998 87:7968). Table 20.4 presents hybrids incorporating epitopes of Table 20.2. Table 20.5 presents Ii-Key/antigenic epitope hybrids incorporating epitopes from peptides of Table 20.3. Table 20.6 presents the deduced amino acids sequence of human proteolipid protein 1 (GenBank gi 19923104). Table 20.7 presents MHC Class II-presented epitopes of proteolipid protein 1 predicted with the SYFPEITHI program. Table 20.8 presents sequences identified experimentally to contain proteolipid protein 1 MHC Class II-presented epitopes. Table 20.9 presents hybrids incorporating epitopes of Table 20.7. Table 20.10 presents Ii-Key/antigenic epitope hybrids incorporating epitopes within peptides of Table 20.8. Table 20.11 presents the deduced amino acids sequence of human myelin-oligodendrocyte glycoprotein precursor (GenBank gi: 2497312). Table 20.12 presents MHC Class II-presented epitopes of oligodendrocyte glycoprotein precursor predicted with the SYFPEITHI program. Table 20.13 presents sequences identified experimentally to contain oligodendrocyte glycoprotein precursor MHC Class II-presented epitopes. Table 20.14 presents hybrids incorporating epitopes of Table 20.12. Table 20.15 presents Ii-Key/antigenic epitope hybrids incorporating epitopes within peptides of Table 20.13.
TABLE-US-00120 TABLE 20.1 Deduced amino acids sequence of human myelin basic protein. (SEQ ID NO: 824) 1 mgnhagkrel naekastnse tnrgesekkr nlgelsrtts ednevfgead 51 anqnngtssq dtavtdskrt adpknawqda hpadpgsrph lirlfsrdap 101 gredntfkdr psesdelqti qedsaatses ldvmasqkrp sqrhgskyla 151 tastmdharh gflprhrdtg ildsigrffg gdrgapkrgs gkdshhpart 201 ahygslpqks hgrtqdenpv vhffknivtp rtpppsqgkg rglslsrfsw 251 gaegqrpgfg yggrasdyks ahkgfkgvda qgtlskifkl ggrdsrsgsp 301 marr
TABLE-US-00121 TABLE 20.2 Predicted MHC Class II-presented epitopes of human myelin basic protein. Ii- SEQ ID PEPTIDE Pos. Sequence Allele Key NO: 20.2.1 284 LSKIFKLGG 01, 11(281) 4 825 20.2.2 88 RPHLIRLFS 01, 03(89), 04(88) 0 826 20.2.3 4 HAGKRELNA 01 0 827 20.2.4 272 HKGFKGVDA 01, 02(273) 0 828 20.2.5 117 LQTIQEDSA 03 0 829 20.2.6 167 RDTGILDSI 03, 11(169) 3 830 20.2.7 66 DSKRTADPK 03 0 831 20.2.8 221 VHFFKNIVT 04 0 832 20.2.9 152 ASTMDHARH 04 0 833 20.2.10 29 KRNLGELSR 01, 04, 11 0 834 20.2.11 176 GRFFGGDRG 04, 11(175) 9 835
Pos. is the first amino acid of the predicted MHC Class II-presented epitope of the specified sequence. Score is the score calculated by the SYFPEITHI program for the first of the given HLA-DRB*--01 alleles which were examined. The second listed allele is for exactly the same epitope or for an overlapping epitope for which the first amino acid position is given in parentheses.
TABLE-US-00122 TABLE 20.3 Experimentally determined MHC Class II- presented epitopes of human myelin basic pro- tein. (SEQ ID NOS 836-841 respectively, in order of appearance) Pre- Pep- Presenting dicted tide Pos. MHC II Sequence epitope 20.3.1 1-44 DR2a MGNHAGKREL NAEKASTNSE TNRGESEKKR NLGELSRTTS EDNE 20.3.2 76-91 DR2a AWQDA HPADPGSRPH LIRLFSRDAP GREDNTFKDR P 20.3.3 131-145 DR2a LDVMASQKRP SQRHG 132 20.3.4 139-153 DR2a; DR1 P SQRHGSKYLA 148 TASTM 20.3.5 80-99 DR2b A HPADPGSRPH 91, 92 LIRLFSRDA 20.3.6 148-162 DR2b LA TASTMDHARH GF 148
Pos. is the first and last amino acids of the segments of hMBP reported to contain MHC Class II-presented epitopes. Sequence is a peptide identified by Pette and colleagues to contain hMBP MHC Class II-presented epitopes (Pette M. Proc Natl. Acad Sci USA. 1998 87:7968). The peptides MBP85-99, MBP85-96 and MBP83-97 have also been characterized by others (Krogsgaard M. J Experi Med. 2000 191:1395-412; Gauthier L. Proc. Natl. Acad Sci USA. 1998 95:11828-33). Presenting allele includes MHC Class II alleles which are reported to present epitopes in the respective segments. Seq. is the sequence of the segment. Predicted epitope is the first amino acid of the epitopes predicted to be presented by respective MHC Class II alleles, using the ProPred algorithm. HMBP(91-98; FIRLFSRDA) (SEQ ID NO: 842) presented by HLA-DRB*0101, 1101, and 1301. hMBP(92-99; IRLFSRDAP) (SEQ ID NO: 843) presented by HLA-DRB*1301 and 1501. hMBP(120-128; IQWDSAATA) (SEQ ID NO: 844) presented by HLA-DRB*03. hMBP(133-141; VMASQKRPS) (SEQ ID NO: 845) presented by HLA-DRB*0101, and 1301. hMPB(148-157; LATASTMDH) (SEQ ID NO: 846) presented by HLA-DRB*0401 and 1101. hMBP(162-170) presented by HLA-DRB*0801.
TABLE-US-00123 TABLE 20.4 Ii-Key/human MBP antigenic epitope hybrids with MHC Class II-Presented epitopes of Table 20.2. (SEQ ID NOS 847-851 respectively, in order of appearance) PEPTIDE Pos. Sequence 20.4.1 284 Ac-LRMK-LSKIFKLGG-NH2 20.4.2 88 Ac-LRMK-RPHLIRLFS-NH2 20.4.3 4 Ac-LRMK-HAGKRELNA-NH2 20.4.4 272 Ac-LRMK-HKGFKGVDA-NH2 20.4.5 117 Ac-LRMK-HKGFKGVDA-NH2
TABLE-US-00124 TABLE 20.5 Ii-Key/human MBP antigenic epitope hybrids with MHC Class II-Presented epitopes of Table 20.3. (SEQ ID NOS 852-855 respectively, in order of appearance) PEPTIDE Pos. Sequence 20.5.1 91 Ac-LRMK-FIRLFSRDA-NH2 20.5.2 92 Ac-LRMK-IRLFSRDAP-NH2 20.5.3 133 Ac-LRMK-VMASQKRPS-NH2 20.5.4 148 Ac-LRMK-LATASTMDH-NH2
[0287]Another major component of CNS myelin, the proteolipid protein (PLP), induces an acute form of EAE in SJL/J mice (Tuohy V K. J Immunol. 1989 142:1523-7; Greer J M. J Immunol. 1992 149:783-8). A principal MHC Class II-presented epitope was found in 139-154 HCLGKWLGHPDKFVGI (SEQ ID NO: 856), and in certain serine-substituted homologs. The sequence of the homologous human sequences are presented in Table 20.2. A second peptide murine 178-191 of PLP (Human homolog sequence: FNT 181 WTTCDSIAFP S) (SEQ ID NO: 857) was also identified to be encephalitogenic in SJL/J (h-2s) mice (Greer J M. J Immunol. 192 149:783-8).
TABLE-US-00125 TABLE 20.6 Deduced amino acids sequence of human proteolipid protein. (SEQ ID NO: 858) 1 mglleccarc lvgapfaslv atglcffgva lfcgcgheal tgtekliety 51 fsknyqdyey linvihafqy viygtasfff lygalllaeg fyttgavrqi 101 fgdyktticg kglsatvtgg qkgrgsrgqh qahslervch clgkwlghpd 151 kfvgityalt vvwllvfacs avpvyiyfnt wttcdsiafp sktsasigsl 201 cadarmygvl pwiafpgkvc gsnllsickt aefqmtfhlf iaafvgaaat 251 lvslltfmia atynfavlkl mgrgtkf
TABLE-US-00126 TABLE 20.7 Predicted MHC Class II-presented epitopes of human myelin proteolipid protein. SEQ Ii- ID PEPTIDE Pos. Sequence Allele Key NO: 20.6.1 77 FFFLYGALL 01, 07(78), 08(78), 8 859 15(78) 20.6.2 243 FVGAAATLV 01, 04(244), 11(244) 4 860 20.6.3 236 FHLFIAAFV 01, 07(232), 11(232) 7 861 20.6.4 250 LVSLLTFMI 01, 03, 04, 13, 15 8 862 20.6.5 162 WLLVFACSA 01, 03(160), 7 863 07(156), 08, 11(160) 20.6.6 99 IFGDYKTTI 03 0 864 20.6.7 199 LCADARMYG 03 0 865 20.6.8 70 VIYGTASFF 03, 04(69), 08(69), 3 866 11(69), 13(69) 20.6.10 157 YEYLINVIH 04 8 867 20.6.11 152 VGITYALTV 08 6 868
Pos. is the first amino acid of the predicted MHC Class II-presented epitope of the specified sequence. Score is the score calculated by the ProPred program for the first of the given HLA-DRB*--01 alleles which were examined. The second listed allele is for exactly the same epitope or for an overlapping epitope for which the first amino acid position is given in parentheses).
TABLE-US-00127 TABLE 20.8 Experimentally determined MHC Class II-pre- sented epitopes of human myelin proteolipid protein. Ii- SEQ ID PEPTIDE Pos. Sequence Key NO: 20.7.1 M139-151 139-154 HCLGKWLGHPDKFVGI 5 869
A series of 4 or more overlapping sequences from position 152 (FVGITYALTVVWLLVFAC) (SEQ ID NO: 870) are presented by alleles HLA-DRB 01, 03, 04, 07, 08, 11, 13, 15. The Ii-Key motif LGKWL (SEQ ID NO: 871) is separated by 5 amino acids from F152.
TABLE-US-00128 TABLE 20.9 Ii-Key/PLP epitope hybrids containing MHC Class II-presented epitopes of Table 20.7. (SEQ ID NOS 872-875 respectively, in order of appearance) PEPTIDE Pos. Sequence 20.8.1 77 Ac-LRMK-FFFLYGALL-NH2 20.8.2 243 Ac-LRMK-FVGAAATLV-NH2 20.8.3 236 Ac-LRMK-FHLFIAAFV-NH2 20.8.4 250 Ac-LRMK-LVSLLTFMI-NH2
TABLE-US-00129 TABLE 20.10 Ii-Key/PLP epitope hybrids containing MHC Class II-presented epitopes of Table 20.8. (SEQ ID NO: 876) PEPTIDE Pos. Sequence 20.9.1 152 Ac-LRMK-FVGITYALTVVWLLVFAC-NH2
[0288]A third protein of myelin, human myelin-oligodendrocyte glycoprotein (MOG) has also been shown to be encephalitogenic in mice (Mendel I. Eur J Immunol. 1995 25:1951-9; Kaye J F. J Neuroimmunol. 2000 102:189-98).
TABLE-US-00130 TABLE 20.11 Deduced amino acid sequence of myelin- oligodendrocyte glycoprotein precursor. (SEQ ID NO: 877) 1 maslsrpslp sclcsfllll llqvsssyag qfrvigprhp iralvgdeve 51 lpcrispgkn atgmevgwyr ppfsrvvhly rngkdqdgdq apeyrgrtel 101 lkdaigegkv tlrirnvrfs deggftcffr dhsyqeeaam elkvedpfyw 151 vspgvlvlla vlpvlllqit vglvflclqy rlrgklraei enlhrtfdph 201 flrvpcwkit lfvivpvlgp lvaliicynw lhrrlagqfl eelrnpf
TABLE-US-00131 TABLE 20.12 Predicted MHC Class II-presented epitopes of human myelin myelin-oligodendrocyte glyco- protein precursor. SEQ Ii- ID PEPTIDE Pos. Sequence Allele Key NO: 20.11.1 155 LVLLAVLPV 01, 03, 04 5 878 20.11.2 200 FLRVPCWKI 01, 07 3 879 20.11.3 31 FRVIGPRHP 01 0 880 20.11.4 217 LGPLVALII 01 5 881 20.11.5 211 FVIVPVLGP 01, 04, 11 6 882 20.11.6 15 FLLLLLLQV 01, 03(18), 04(18), 0 883 15(12) 20.11.7 43 LVGDEVELP 03 5 884 20.11.8 99 LLKDAIGEG 03 0 885 20.11.9 111 LRIRNVRFS 03, 08 6 886 20.11.10 164 LLLQITVGL 04, 07(166) 0 887 20.11.11 149 WVSPGVLVL 07 5 888 20.11.12 179 YRLRGKLRA 08 0 889 20.11.13 229 WLHRRLAGQ 08 0 890
Pos. is the first amino acid of the predicted MHC Class II-presented epitope of the specified sequence. Score is the score calculated by the ProPred program for the first of the given HLA-DRB*--01 alleles which were examined. The second listed allele is for exactly the same epitope or for an overlapping epitope for which the first amino acid position is given in parentheses).
TABLE-US-00132 TABLE 20.13 Experimentally determined MHC Class II- presented epitopes of myelin-oligodendrocyte glycoprotein precursor. PEPTIDE Pos. Sequence SEQ ID NO: 20.12.1 h21-39 LLQVSSSYAG QFRVIGPRH 891
TABLE-US-00133 TABLE 20.14 Ii-Key/MOG epitope hybrids containing MHC Class II-presented epitopes of Table 20.12. (SEQ ID NOS 892-895 respectively, in order of appearance) PEPTIDE Pos. Sequence 20.13.1 155 Ac-LRMK-LVLLAVLPV-NH2 20.13.2 200 Ac-LRMK-FLRVPCWKI-NH2 20.13.3 31 Ac-LRMK-FRVIGPRHP-NH2 20.13.4 211 Ac-LRMK-FVIVPVLGP-NH2
TABLE-US-00134 TABLE 20.15 Ii-Key/MOG epitope hybrids containing MHC Class II-presented epitopes of Table 20.13. (SEQ ID NOS 896 & 897) PEPTIDE Pos. Sequence 20.14.1 16-27 Ac-LRMK-FLLLLLLQVSSSY-NH2 20.14.2 13-23 Ac-LRMK-FRVIGPRHPIRA-NH2
Peptide 20.14.1 contains three overlapping MHC Class II-presented epitopes presented by alleles HLA-DRB 01, 03, 04, 07, 11, 13, and 15. Peptide 20.14.2 contains two overlapping MHC Class II-presented epitopes presented by alleles HLA_DRB 01, 08 and 11.
Example 21
Identification and Use of Peptide Sequences Containing Ii-Key Motifs Appropriately Placed from the N-Terminal End of MHC Class II Antigenic Epitopes
[0289]In another aspect this invention relates to methods to select a preferred set of biologically active MHC Class II-presented epitopes in antigenic proteins. Specifically, this disclosure provides methods to identify in the amino acid sequence of a protein the presence or absence of a Ii-Key immunoregulatory motif of 5 amino acids preceding an experimentally determined or algorithm-predicted, MHC Class II-presented, antigenic epitope. This immunoregulatory Ii-Key motif enhances charging of the linked antigenic epitope into the antigenic peptide binding site of MHC Class II molecules. Given predictions of antigenic epitopes within a protein, identifying the subset of those epitopes preceded by an Ii-Key motif improves greatly the efficiency of vaccine peptide selection. Also, by modifying the sequence of a protein, either to introduce or to eliminate an Ii-Key motif before selected MHC Class II-presented epitopes, the immunological response to that protein can be altered.
[0290]This disclosure presents a method to identify an Ii-Key immunoregulatory motif. Specifically, in the sequence of a protein, the immunoregulatory, Ii-Key motif is a segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, where that contiguous 5 amino acid segment is separated by 5 to 11 amino acids from the N-terminal residue of the MHC Class II-presented epitope. The subset of such antigenic epitopes with the presence of an appropriately spaced Ii-Key motif are more potent than epitopes not preceded by such an Ii-Key motif in enhancing the potency of the CD4+ T cell immune response. Such epitopes are also more likely to be dominant or biologically active. Peptides with such epitopes are favored as vaccines to protect against infectious diseases and cancer, and as immunosuppressive vaccines for allergy, autoimmune disease, and graft rejection.
[0291]In another aspect, this invention relates to therapeutic proteins with sequences which are modified in a manner to alter immune responses to the therapeutic proteins. Such proteins include therapeutic proteins, such as hormones, cytokines, or other molecules interacting with cell surface receptors, and enzymes. Modifications of an Ii-Key motif can be made to eliminate its function, or such an Ii-Key motif can be introduced before a putative antigenic epitope when such a motif is lacking. Such modifications can suppress a disadvantageous immune response to the therapeutic protein. Such products include the therapeutic protein, and fragments thereof, and genetic constructs leading to their expression.
[0292]This invention is based in the discovery that a naturally occurring Ii-Key motif appropriately spaced before a potential antigenic epitope, selects for biological activity in a subset of MHC Class II binding motifs. The binding of radiolabeled, photo-crosslinking, antigenic peptides to MHC Class II molecules is more efficient during the cleavage and release of the Ii protein from MHC Class II alpha and beta chains in the presence of cathepsin B but not cathepsin D (Daibata M. Mol Immunol. 1994 31:255-260). Mutants of putative cleavage sites in the Ii protein confirmed the role of residues in the R78-K86 region in the final cleavage and release of the avidin-labeled Ii fragments that are still immunoprecipitated with MHC Class II alpha and beta chains (Xu M. Mol Immunol. 1994 31:723-731). The biochemical mechanism of this "final cleavage" region was tested with synthetic Ii-L87-K, which contains six residues with cationic side chains, no anionic side chains and four spaced prolines. This Ii-Key peptide promoted binding or release of antigenic peptides in vitro (Adams S. Eur J Immunol. 1995 25:1693-1702). Structure-activity relationships were characterized with 160 homologs, in antigenic peptide presentation to murine T hybridomas (Adams S. Arzneimittel-Forschung. 1997 47:1069-1077), and with purified HLA-DR1 in a peptide binding/release assay (Xu M. Arzneimittel-Forschung. 1999 49:791-9). The Ii-Key segment hIi(77-92) of the Ii protein promotes the binding of synthetic peptides to MHC Class II molecules by acting at an allosteric site adjacent to one end of the antigenic peptide-binding trough. Furthermore, by coupling this Ii-Key segment through a simple polymethylene linker to an antigenic peptide, the potency of presentation of the epitope to a T hybridoma was enhanced 500 times, relative to only the antigenic epitope (Humphreys R E. Vaccine. 2000 18:2693-7). Thus, comparable, naturally occurring, appropriately spaced Ii-Key motifs can be expected to promote the selection of the subset of antigenic epitopes before which they occur at an appropriately spaced interval. Since synthetic hybrids containing linkers of either the natural sequence of Ii-protein extending from the LRMK (SEQ ID NO: 3) motif, or 5-amino-pentanoic acid, were comparably active, no specific side chain interactions were required between the linker and the alpha helices of the antigen-binding site. Thus, the specific amino acids forming a spacer region appear not to be relevant. The hypothesis that naturally occurring Ii-Key-spacer motifs regulate selection of potential MHC Class II epitopes in vivo was tested for presence and spacing of a generic Ii-Key motif from both the N-terminus (active hypothesis) or the C-terminus (indifferent hypothesis) of defined MHC Class II-presented epitopes.
[0293]Ii-key motifs in antigenic proteins serve to catalyze insertion of closely following, MHC Class II-presented, antigenic epitopes into peptide binding sites of MHC Class II molecules. It is the Ii-Key motif appropriately spaced before a potential MHC Class II binding peptide, which makes that epitope immunogenic. From this discovery, comes a novel method to select a subset of epitopes identified by consensus motifs for their dominant role in antigen presentation. Such epitopes can be exploited in preventative and therapeutic vaccines. Therapeutic proteins can also be modified to either enhance or suppress immunogenicity.
[0294]The following method to identify Ii-Key motifs within the amino acid sequence of antigenic proteins was designed prior to examination of the data set, and tested without alteration. An Ii-Key box was defined to be 5 contiguous residue positions containing at least two residues of the group L, I, V, F, and M and at least one residue of the group H, K, and R. This was the simplest model based on two concepts.
[0295]The first critical concept in the design of the method of this invention was the discovery of the motif defining the Ii-Key "core segment". In the natural sequence of the human Ii protein, the core motif was defined to be L77RMK (SEQ ID NO: 3) on the basis of previous experimental studies showing retention by this peptide of at least half-maximal activity of the best Ii-Key peptide in a systematic series of hybrids (Adams S. Arzneimittel-Forschung. 1997 47:1069-77; Humphreys R E. Vaccine 2000 18:2693-7). The core motif of four amino acids is contained within a previously defined segment of 7 amino acids (LRMKLPK) (SEQ ID NO: 4) studied through analysis of 84 homologs with 12 amino acid substitutions at each residue position (Adams S. Arzneimittel-Forschung. 1997 47:1069-77; Xu M. Arzneimittel-Forschung. 1999 49:791-9). In those studies, biological activity was discovered not to require precisely alternating hydrophobic/cationic side chains, provided that at least two hydrophobic and one cationic residue were present within a segment of 4 or 5 residues. For this reason, in defining the structure of an Ii-Key motif in the present invention, the presence of two hydrophobic side chains and at least one cationic side chain in any sequence within a stretch of 5 amino acids was considered to be sufficient for the function of Ii-Key in charging antigenic epitopes into MHC Class II molecules.
[0296]The second critical concept in the design of the method of this invention was the discovery of the functional equivalence of L, I, V, F, and M in one set, and H, K, and R in another respective set, in governing the structure of locally folded segments of proteins. This equivalency was discovered in a systematic survey of groups of amino acids in certain patterns either throughout proteins (Vazquez S. Proc Natl Acad Sci USA. 1993 90:9100-4) or in geometrically defined positions around alpha helices (Torgerson S. J Biol Chem. 1991 266:5521-24; Vazquez S. J Biol Chem. 1992 267:7406-10; Rennell D. J Biol Chem. 1992 267:17748-52).
[0297]The above method of identifying naturally occurring Ii-Key motifs appropriately spaced from the N-terminus of MHC Class II-presented epitopes was validated though the following analysis. All 36 of the antigenic epitopes reported by Rammensee and colleagues (Rammensee H G. Immunogenetics. 1995 41:178-228) in their analysis of motifs predicting MHC Class II-presented peptides were analyzed, excluding homologous epitopes (for example from difference MHC Class II alleles). The sequences for each of these reported proteins upstream and downstream with respect to the antigenic epitope were obtained from GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein). Given the definition of an Ii-Key box to contain 5 contiguous residue positions containing at least two residues of the group L, I, V, F, and M and at least one residue of the group H, K, and R, then the distance of such boxes from the N-terminus or C-terminus of known MHC Class II-presented epitopes within antigenic proteins were determined. A significant minimal spacing of such Ii-Key boxes from the N-terminus but not the C-terminus of such epitopes was anticipated because the biological effect was anticipated to be at the N-terminus and not the C-terminus. The predicted lack of biological effect of such boxes at the C-terminus (and thus the spacing form the C-terminus) was a useful null hypothesis for statistical analysis. Segments of 5 contiguous residues extending progressively in an N-terminal direction from a Rammensee-reported antigenic epitope were tested for the occurrence of an Ii-Key box. The box with the least number of intervening residue positions between the Ii-Key box and the Rammensee-reported antigenic epitope was scored in terms of the number of intervening residue positions (Table 1). A similar analysis, in the mirror image manner was scored distally from the C-terminus of the Rammensee-reported antigenic epitope. The least intervening residue positions to the first Ii-Key box were scored from the C-terminus of the antigenic epitope (Table 21.1).
[0298]The response of T cells to a MHC Class II-restricted antigenic epitope is enhanced greatly when that epitope is presented to T cells in a synthetic hybrid peptide linking an Ii-Key sequence (such as Acetyl-Leu-Arg-Met-Lys (SEQ ID NO: 3) [Ac-LRMK-](SEQ ID NO: 3)) thorough a spacer 5-amino-pentanoic acid to the N-terminus of the MHC Class II-presented epitope. In order to test whether homologous, naturally occurring, Ii-Key/spacer/epitope motifs select for antigenic epitopes during processing of antigenic proteins, the frequency of one prototypic pattern was tested in a series of proteins with respect to placement about their experimentally determined MHC Class II epitopes. A non-empirical, N-terminal distribution (p<0.025) was found for a prototypic Ii-Key/spacer pattern (a 5 amino acid segment containing at least two residues of the group Leu, Ile, Val, Phe, and Met [LIVFM] (SEQ ID NO: 790) and at least one of the group Arg, His, and Lys [RHK]), and spacer length of 4 to 8 residues. This placement was significant in comparison to the empirical placement of the motif from the C-terminus (the indifferent hypothesis). This observation helps to explain the biological activity of only some epitopes among sets of predicted "MHC Class II consensus motifs" in antigenic proteins. It also leads to methods to modulate the immune response to certain antigens.
TABLE-US-00135 TABLE 21.1 Spacer length (residue positions) between Ii-Key boxes and terminus of the antigenic epitope. Spacer length N-terminus C-terminus 0 2 9 1 1 2 2 1 3 3 0 0 4 0 0 5 2 3 6 5 1 7 0 2 8 1 2 9 4 1 10 4 1 11 2 2 12 0 1 13 2 0 14 2 0 No box 9 7 Sum 29 29
[0299]By inspection, the pattern for occurrence and placement of the Ii-Key box from the N-terminus of the epitope is very different from that on the C-terminal side, and the pattern of the C-terminal side is not different from that expected to be generated by the Monte Carlo model (random assignment of residue types at the frequency of occurrence in the data set). These observations were subjected to quantitative statistical analysis. The probabilities that the observed difference might have occurred as a random event for various groupings of spacer lengths are given in Table 21.2.
TABLE-US-00136 TABLE 21.2 Analysis of distributions of Ii-Key motifs of certain spacer lengths. Spacer Chi square length N(obs) C (obs) Vs 0-4 p≦ 0 to 4 4 14 5 to 8 8 6 4.097 0.05 5 to 11 18 12 6.467 0.025 5 to 14 22 13 7.854 0.01
[0300]Variations on the definition of a positive Ii-Key box and length and character of a spacer can be proposed. However, the testing of such alternatives must be performed on a new data set, to avoid a type I statistical error. Such an error did not occur in our present study since the scored motif was precisely and completely defined prior to examination of the current data set. We refrain from the suggestion of alternative Ii-Key box and spacer patterns, which might fit this data set better.
[0301]The fact that Ii-Key motif are found appropriately spaced to the N-terminal side of antigenically active MHC Class II-epitopes supports the view that such Ii-Key motifs are active in the selection of peptides to become bound to MHC Class II molecules in post-Golgi antigen charging compartment of antigen presenting cells. Likewise, the fact that Ii-Key/MHC Class II antigenic epitope hybrid peptides are well presented after immunization in Freund's incomplete adjuvant indicates that the Ii-Key motif on such peptides is active in selection of the epitopes of those peptides for charging in the post-Golgi antigenic peptide charging compartment.
[0302]Alteration can be introduced in therapeutic proteins to enhance a favorable characteristic. Use of many therapeutic proteins is limited because an immune response, as evidenced by neutralizing antibodies, developed against the protein, as has been observed with insulin, erythropoietin, and beta-interferon. Given a therapeutic protein to which an immunological response in some patients limits therapeutic use, the current invention may be used to prevent the immune response or mute the response in patients who have developed such response. The process includes the following steps. Examine the primary amino acid sequence. Define within the primary amino acid sequence motifs of MHC Class II-restricted epitopes. Choose epitope which are suitable to be altered at a few amino acids, in a manner to create an Ii-Key box-spacer motif N-terminal to the first residue of the antigenic motif. Synthesize the protein by recombinant molecular genetic methods or by peptide synthesis method. Test the synthetic variant for the induction of a suppressing immune response to the protein, for example by reduction of antibodies upon challenge with the parental therapeutic protein.
[0303]Ii-Key motifs upstream to selected MHC Class II-presented epitopes in clinically relevant antigens might lead to novel therapeutic vaccines. Perhaps engineering an Ii-Key spacer motif can alter the immunogenicity of an antigenic epitope within a protein. Particularly for the most N-terminal antigenic epitopes, introduction of one or a few altered residues may be tolerated (Rennell D. J Biol Chem. 1992 267:17748-52). Such manipulations could generate forms of therapeutic proteins amenable to induction of tolerance.
[0304]This method of analysis can be extended to additional antigenic or therapeutic proteins of interest to which in vivo immunosuppressive responses are damaging to the host. For example, an examination of antigenic epitopes in HIV reverse transcriptase shows that antigenic epitopes there may have such upstream Ii-Key like segments, perhaps governing biological potency to establish dominance in establishing tolerance. A conserved universal Th epitope in HIV-1 reverse transcriptase is preceded by an Ii-key-spacer motif. An HIV-1 reverse transcriptase epitope, which was highly conserved among various HIV-1 isolates and was presented by at least four HLA-DR molecules, was discovered by Van der Burg and colleagues in a systematic survey of 20 amino acid peptides through the sequence of that enzyme (van der Burg. J Immunol. 1999 162:152-160). This peptide and the upstream 15 amino acids are the following:
TABLE-US-00137 271-290 NDIQK LVGKL NWASQI YPGIKVRQLCKLLRGTKALT (SEQ ID NO: 898) (SEQ ID NO: 899)
[0305]Possible MHC Class II-presented epitopes are single underlined and the putative Ii-Key motifs is double underlined. This example illustrates how the presence of an Ii-Key motif appropriately spaced before a MHC Class II-presented epitope can enhance the presentation of that epitope. Further, the presence of the Ii-Key motif may be responsible for the development of a highly efficient immunosuppressive response.
Example 22
Enhancement of Antibody, T Helper Cell, and CTL Responses to MHC Class I Epitopes by Immunizations with Ii-Key/MHC Class II Epitope Hybrids
[0306]Substantially greater immune responses were found in mice immunized with epitopes presented in Ii-Key antigenic epitope hybrids, than in antigenic epitope peptides alone. The immune responses were measured by titers of antibodies to individual antigenic epitopes, epitope-specific-CD4+/IFN-γ+ cells, and epitope-specific IFN-γ release in the ELISPOT assay.
[0307]Two different antigenic epitopes, from pigeon cytochrome C and from HIV gp160, were used in these comparative studies. The PGCC(95-104) epitope was presented in an Ii-Key/antigenic epitope hybrid peptide (Ac-LRMK-ava-IAYLKQATAK-NH2; "Ii-Key/PGCC"; SEQ ID NO: 900) or in an antigenic epitope peptide (Ac-IAYLKQATAK-NH2; ("PGCC"); SEQ ID NO: 901). The HIV gp160(843-855) epitope was presented in: 1) an Ii-Key/antigenic epitope hybrid peptide with two ava residues (Ac-LRMK-ava-ava-AYRAIRHIPR-NH2; "Ii-Key/two-ava/gp160(843-855)"; SEQ ID NO: 902); 2) an Ii-Key/antigenic epitope hybrid peptide with one ava residue (Ac-LRMK-ava-AYRAIRHIPR-NH2; "Ii-Key/one-ava/gp160(843-855)"; SEQ ID NO: 903); 3) an Ii-Key/antigenic epitope hybrid peptide with one ava residue (Ac-LRMK-ava-YRAIRHIPR-NH2; alanine-843 is deleted for more precise measurement of space between epitope and Ii-Key; "Ii-Key/one-ava/gp160(844-855)"; SEQ ID NO: 904); 4) an Ii-Key/antigenic epitope peptide with no "ava" (Ac-LRMK-AYRAIRHIPR-NH2; "gp160(843-855)"); SEQ ID NO: 905). "ava" is delta-aminovaleric acid, which is 5-aminopentanoic acid. Its maximal linear extent approximates the length of the backbone atoms of 2.5 amino acids in a peptidyl sequence. Thus, the two-ava linker bridges the Ii-Key motif from the antigenic epitope by about 5 amino acids. Five amino acids is the number of amino acids of an extended antigenic peptide occupying the antigenic peptide binding trough from the residue that lies in the P1 site to the N-terminally exposed end of a peptide that lies in that trough.
[0308]An ELISA assay for antibody responses following immunization with the experimental peptides indicated above was performed as follows. Fifty microliters (μl) of a solution of the coating peptide at 2 μg/well in 0.1 M carbonate buffer, pH 9.5 was added to each well of a 96-well Nunc-immunoplate (#442404) for an overnight incubation at 4° C. After aspiration, 250 μl of phosphate-buffer saline solution containing 3% fetal bovine serum (assay diluent) was added to each well for 2 hr at RT. After washing three times with assay diluent, 50 μl of 20-times diluted mouse serum in 1:3 serial dilution in assay diluent was added to each well for 2 hr at RT. After washing three times with assay diluent, 50 μl of 1 μg/ml biotinylated goat anti-mouse IgG1 or IgG2a was added to each well and incubated for 1 hr at RT. After washing three times with assay diluent, 50 μl of streptavidin-horse radish peroxidase conjugate (1:1000) was added to each well and incubated for 30 min at RT. After washing three times with assay diluent, 100 μl of tetramethylbenzidine/H2O2 solution (Pharmingen 264KK) was added to each well for 15 min in the dark at room temperature. The reaction was stopped with 100 μl 1N H2SO4 in each well and the absorbance was read immediately at 450 nm.
[0309]Significantly greater antibody titers against PGCC epitope were induced by immunizing with Ii-Key/PGCC(95-104) than with PGCC(95-104) peptide alone, either in CFA (Table 22.1) or in IFA (Table 22.2). C3H/HeJ mice (H-2k) were immunized with 10 nmole of peptides (50 μl) emulsified with an equal volume of complete Freund's adjuvant (CFA), subcutaneously at the base of the tail. On day 14 the mice were boosted subcutaneously at the base of the tail with 10 nmole of peptides (50 μl) emulsified with an equal volume of incomplete Freund's adjuvant (IFA). On day 28 the mice were boosted intravenously with 40 nmole peptides dissolved in Hank's balanced salts solution (HBSS). On day 33 the mice were sacrificed and serum samples were assayed for antibody titers against the PGCC(95-104) epitope peptide.
TABLE-US-00138 TABLE 22.1 Antibody induction after immunizations with PGCC(95-104) epitope with complete Freund's adjuvant. Dilution-1 Immunogen 20 60 180 540 Ii-Key/PGCC 1.409 1.489 0.252 0.53 PGCC 0.128 0.057 0.016 0.004 None 0.105 0.72 0.049 0.036
[0310]To vaccinate with IFA, C3H/HeJ mice (H-2k) were immunized with 10 nmole of peptides (50 μl) emulsified with equal amount of IFA, subcutaneously at the base of the tail. On day 14 the mice were boosted with 10 nmole of peptides (50 μl) emulsified with an equal volume of IFA, again subcutaneously at the base of the tail. On day 28 the mice were boosted intravenously with 40 nmole peptides dissolved in Hank's balanced salts solution (HBSS). On day 33 the mice were sacrificed and serum samples were assayed for antibody titers against the PGCC(95-104) epitope peptide.
TABLE-US-00139 TABLE 22.2 Antibody induction after immunizations with PGCC(95-104) epitope with incomplete Freund's adjuvant. Dilution-1 Immunogen 20 60 180 540 1620 4860 Ii-Key/PGCC 3.503 2.995 0.782 0.205 0.071 0.024 PGCC 0.102 0.186 0.019 0.005 -0.003 0.003 None 0.042 0.004 -0.003 0.007 0.006 0.005
[0311]Significantly greater antibody titers against the HIV gp160(843-855) epitope resulted from immunization with Ii-Key/HIV gp160(843-855) hybrid than with HIV gp160(843-855) peptide, both being administered in saline solution (Table 3). B10.A (5R) mice (H-2k/b) were immunized with 20 nmole of peptides in 50 μl phosphate-buffered saline solution, intramuscularly in right and left rear legs on days 1 and 2, respectively. On day 14 the mice were boosted intramuscularly with 40 nmole of peptides in 200 μl Hank's balanced salts solution intramuscularly in a rear leg. On day 30 the mice were boosted intravenously with 40 nmole peptides dissolved in Hank's balanced salts solution (HBSS). On day 35 the mice were sacrificed and serum samples were assayed for antibody titers against the HIV gp160(843-855) peptide.
TABLE-US-00140 TABLE 22.3 Antibody induction after immunizations with HIV gp160(843-855) epitope peptide in saline solution. Dilution-1 Immunogen 20 60 180 540 Ii-Key/one-ava/ 0.600 0.157 0.073 0.024 gp160(843-855) Ii-Key/two-ava/ 0.131 0.039 0.027 0.003 gp160(843-855) Gp160(843-855) 0.052 0.023 0.000 -0.005 None 0.084 0.045 0.004 -0.003
[0312]The above results demonstrate that presentation of the antigenic epitope in an Ii-Key/antigenic epitope hybrid greatly enhances induction of an antibody response regardless whether CFA or IFA is the vehicle for the first immunization. IFA is composed of bayol oil, and CFA is composed of IFA to which has been added heat-killed mycobacterium tuberculosis. In the experiments of Tables 1 and 2, IFA was the vehicle for the second immunization, a subcutaneous booster injection, and HBSS was the vehicle for the third immunization, an intravenous booster injection. Because CFA and IFA mediate phagocytosis of the peptides by professional antigen presenting cells, their use leads to charging of the epitope peptide in the post-Golgi, antigen charging compartment. Therefore, Ii-Key/antigenic epitope hybrids have benefit from two mechanisms for charging to MHC Class II molecules: at cell surface MHC Class II molecules, for example on paraformaldehyde-fixed cells (Adams S. Eur J. Immunol 1995 25:1693-1702), and in the post-Golgi antigen charging compartment, after internalization.
[0313]The frequency of CD4.sup.+/IFN-γ.sup.+ Th-1 helper T cells were greatly increased after immunization with Ii-Key/gp160(843-855) hybrid, as compared to immunization with gp160(843-855) peptide (Table 4). To test the mechanism for the much greater immunogenecity of Ii-Key/epitope hybrids epitope peptides, B10(A) 5R mice were immunized with 10 nmole of either gp160(843-855) peptide or Ii-Key/gp160(843-855) in CFA subcutaneously at the right side of the base of the tail. On day 10 the mice were boosted with 40 nmole of hybrid peptide or epitope peptide in saline by intravenous injection. On day 26 mice were sacrificed and splenic cells were obtained. 1×106 splenic mononuclear cells were stimulated overnight in the presence of 10 units of recombinant IL-2 and indicated peptides (10 μg/ml). During the last 3 hours of incubation, 2 μM monensin (Golgi-stop, Pharmingen) was added to the cultures, and cells were then stained for both cellular surface markers and intracellular IFN-γ. The FACS assay to quantify antigen specific Th1 helper cell responses was performed as follows. The cells were incubated with 1 μg fluorescein blocking reagent (FC/block, Pharmingen) per 106 cells in 100 μl of staining buffer (Dulbecco's phosphate-buffered saline solution without magnesium or calcium). Those cells were stained at 106 cells/100 μl with either rat-anti-mouse CD3 or CD4 monoclonal antibodies for 30 min at 4° C. After washing, the cells were re-suspended, fixed with 4% formaldehyde, and permeablized with 0.5% saponin for 20 min at 4° C. The cells were suspended in staining buffer with 0.5% saponin, and stained with the appropriate anti-cytokine or isotype control antibody (IFN-γ, XMG1.2, Pharmingen or IFN-γ isotype control, R3-34, Pharmingen). The cells were incubated for 30 min at 4° C. in the dark, washed and fixed for 10 min with 0.3% formaldehyde in 0.5% saponins in staining buffer for flow cytometric analysis. Flow cytometric analysis was performed as follows. First, CD3.sup.+ cells were gated using dual color dot plot of side scatter versus CD3 FITC as the T-gate population. The CD3.sup.+ cells were then analyzed for CD4 expression to target the CD3.sup.+/CD4.sup.+ T-helper cell population. Within this specific cell population, dual color dot plots were used to analyze intracellular interferon-γ cytokine stained by phycoerthyrin (PE)-labeled antibody versus CD3.sup.+/CD4.sup.+ cells stained with fluorescein-labeled antibody.
[0314]The increase in CD4.sup.+/IFN-γ.sup.+ cells is consistent with stimulation and expansion of antigen-specific Th-1 helper T cells. The Th-1 helper T cell subpopulation is characterized by predominant production of IFN-γ while the Th-2 subpopulation of helper T cells is characterized by preferential production of IL-4 and IL-10. Anti-CD3 antibody, which reacts with T cell receptors, measures all T cells, both resting and CD4.sup.+ and CD8.sup.+ subpopulations. The increase from about 1.0% to about 2.0% in the CD4+/IFNγ.sup.+ antigen-specific subpopulation (as compared with naive mice) is consistent with studies of others on mice immunization with various antigens (Caraher E M. J Immunol Methods 2000 244:29; O'Hagan D. J Virol 2001 75:9037; Karulin A Y. J Immunol. 2002 168:545-53; Targoni O S. J Immunol. 2001 166:4757-64; Hesse M D. J Immunol. 2001 167:1353-61; Heeger P S. J Immunol. 2000 165:1278-84; Helms T. J Immunol. 2000 164:3723-32; Yip H C. J Immunol. 1999 162:3942-9).
TABLE-US-00141 TABLE 22.4 Double color FACS analysis of murine splenic T cells after vaccination with Ii-Key/HIV gp160(843-855) hybrid or epitope peptides. Percentage of cells Immunogen CD4.sup.+ CD4.sup.+/IFN-γ.sup.+ CD3.sup.+/IFN-γ.sup.+ Naive 32.40 1.06 2.47 gp160(843-855) 31.03 0.94 2.05 Ii-Key/gp160 31.70 1.83 4.03 (843-855)
[0315]An ELISPOT assay for IFN-γ cytokine responses was performed in order to titer more exactly splenic T lymphocyte subset responses to immunization with Ii-Key-ava-gp160(843-855) or gp160(843-855). The assay was performed as follows. A solution of the cytokine-specific capture antibody (100 μl at 6 μg/ml in phosphate-buffered saline solution, pH 7.2) was added to each well of a 96-well Immunospot plate (M200) for an overnight incubation at 4° C. After aspiration, phosphate-buffer saline solution 200 μl containing 10% fetal bovine serum and 1 penicillin-streptomycin-glutamine (mouse medium) was added to each well for 2 hr at RT. After washing four times with 1% Tween-20 in phosphate-buffered saline solution (wash buffer I), 100 μl of single cell suspensions from the spleens of immunized mice at 106 cells/well were re-stimulated with 100 μl of peptide-epitope at 5 μg/well in mouse medium and incubated for 20-40 hr at 37° C., 5% CO2. After washing twice with phosphate-buffered saline solution (wash buffer II) and four times with wash buffer 1,100 μl of 2 μg/ml biotinylated anti-mouse IFN-γ in 1× phosphate buffer saline with 10% fetal bovine serum (dilution buffer) was added to each well for 2 hr at RT. After washing five times with wash buffer I, 100 l of streptavidin-horse radish peroxidase conjugate (1:500) in dilution buffer was added to each well for 1 hr at RT. After washing four times with wash buffer I and two times with wash buffer II, 100 μl of the 3-amino-9-ethylcarbazole/H2O2 substrate (Pharmingen 551951) was added and incubated for 30-60 min in the dark at RT. The reaction was stopped by washing three times with 200 μl of deionized water. ELISPOT data analysis was performed by using the Immunospot 1.7e software (Cellular Limited Technology). Digitized images of quadruplicate wells were analyzed for the presence of spots in which color density exceeds background based on comparison of control cells (naive splenocytes) and experimental cells (splenocytes of immunized mice) cells. Additional counting parameters for spot size and circularity were applied to gate out speckles caused by non-specific antibody binding. Each spot represents a single cell secreting IFN-γ.
[0316]IFN-γ/Th-1 responses were elicited to the HIV gp160(843-855) epitope from immunization with Ii-key/HIV gp160(844-855) hybrid and HIV gp160(843-855) peptide, both being administered in saline solution (Table 5). C3H/HeJ(H-2k) mice were immunized with 40 nmole of peptides (50 μl) emulsified with equal volume of incomplete Freund's adjuvant (IFA), subcutaneously at the base of the tail. On day 13 the mice were boosted subcutaneously at the base of the tail with 40 nmole of peptides (50 μl) emulsified with equal amount of incomplete Freund's adjuvant (IFA). On day 31, the mice were boosted intravenously with 40 nmole peptides dissolved in 100 μl Hank's balanced salts solution (HBSS). On day 35, the mice were sacrificed and single cell suspensions from spleens were assayed for IFN-γ responses to HIV gp160(843-855) peptide.
TABLE-US-00142 TABLE 22.5 ELISPOT analysis of murine splenic T cells after vaccination with Ii-Key/HIV gp160(843-855) hybrid or epitope peptides. Immunogen Number Size Ii-Key/gp160(844-855) 59 (+/-5) 0.028 (+/-0.006) gp160(843-855) 5 (+/-3.6) 0.019 (+/-0.01) None 0 0
Number is the mean number of spots (and standard deviation) in triplicate wells and Size is the mean spot size in mm2 (and standard deviation). Ii-Key/gp160(844-855) has one ava spacer (Ii-key-ava-gp160(844-855)).
[0317]The data of Tables 22.4 and 22.5 supports the view derived from the data of Tables 1-3 that Ii-Key/antigenic epitope hybrids are significantly more potent immunogens than the comparable antigenic peptides. Furthermore, antigenic epitopes in Ii-Key/antigenic epitope hybrids are well presented after subcutaneous immunization in PBS, without an adjuvant. This fact points to effective use of these peptides as vaccines in humans, for whom various other adjuvants, e.g., CFA or even IFA, are either contraindicated or not preferred.
[0318]A cDNA sequence for an Ii-Key/antigenic epitope hybrid peptide can be constructed for delivery as a minigene DNA vaccine. Such a construct is either a minigene composed of one or several repeated gene constructs each encoding the Ii-Key/antigenic epitope, or as such one or more inserts into a DNA vaccine coding for expression of a protein from which the antigenic epitope of the minigene construct was derived. Standard molecular biology techniques are used to generate such minigene constructs (Leifert J A. Hum Gene Ther. 2001 12:1881-92; Liu W J. Virology. 2000 273:374-82).
[0319]The DNA structure coding for such a minigene contains the codons for 1) a biologically active Ii-Key peptide such as LRMK or a biologically active homolog of the Ii-Key peptide as taught in U.S. Pat. No. 5,919,639, 2) a spacer composed of ala-ala-ala or other biologically accepted functional equivalent of ava or ala-ala-ala, and 3) the antigenic epitope.
[0320]This disclosure reveals that a spacer composed of one delta-aminovaleric acid, which is 5-aminopentanoic acid, is preferred to a spacer composed of two such residues or no spacer at all. Since the linear extent of one delta-aminovaleric acid residue in an Ii-Key/antigenic epitope peptide approximates the linear extent along the backbone of about 2.5 amino acids, the length of the spacer-equivalent in the DNA construct of the minigene is preferably 2, 3 or 4 amino acids, but the length of that spacer can extend from one to 11 amino acids. The codons in the spacer-equivalent segment of the minigene can encode functionally accepted amino acids, but preferable are drawn from the group including small side chain amino acids such as alanine, glycine and serine.
[0321]As with other examples presented in this disclosure, the amino acids of the antigenic epitope segment of the Ii-Key/antigenic epitope hybrid may be composed of amino acid sequences coding for one MHC Class II-presented antigenic epitope only, or for such an epitope with attached or overlapping sequence(s) coding for one or more of the following a) a second MHC Class II-presented epitope, b) a MHC Class I-presented epitope, and c) an antibody-recognized epitope.
[0322]Immunization with Ii-Key/Class II epitope hybrids enhances CTL responses to MHC Class I epitopes activity by augmenting antigen-specific T helper cell responses. Improved potency of MHC Class II epitope presentation potentiates responses to activity of MHC class I epitopes. Mice were immunized with mixtures of Ii-Key/MHC Class II hybrid with CTL epitope, or MHC Class II epitope+CTL epitope, or CTL epitope alone. The ELISPOT assay showed that immunizing mice with Ii-Key/MHC Class II hybrid with CTL epitope produced enhanced CTL activity (Table 22.6). C3D2F1/J mice were immunized subcutaneously at the base of the tail with a mixture of either: 1) 40 nmole of Ii-key/HIV helper T epitope GP120(91-100)] & 20 nmole of HIV CTL epitope (p18) in IFA, 2) 40 nmole of HIV helper T epitope GP120(91-100) and 20 nmole HIV CTL epitope (p18) in IFA, 3) 20 nmole of HIV CTL epitope (p18) in IFA, or 4) No immunogen. On day 14, the mice were boosted with the same immunogens, as described above, at the base of the tail. On day 32, the mice were boosted one more time subcutaneously. Single cell suspensions from individual mouse spleens were challenged ex vivo five days following the last boost in cultures (106 cells/well) containing CTL epitope p18 (5 g/well), a non-specific epitope (5 g/well) and medium alone. Table 22.6 represents the mean spot values and SD calculated from data averaged from three mice per group in six to nine wells.
TABLE-US-00143 TABLE 22.6 ELISPOT analysis of CTL spots after immunization of mice with mixture of Ii-Key/gp120 (91-100) with CTL epitope p18 or mixture of gp120(91-100) with p18, or p18 alone. Non-specific CTL peptide Immunogen reaction reaction Medium Ii-Key/gp120 (91-100) + 27 6 0 p18 Gp120 + p18 11 5 0 P18 7 0 0 naive 5 2 0
[0323]Thus, mice immunized with Ii-Key/MHC Class II helper epitopes+CTL epitope exhibited a much greater antigen specific CTL response than mice immunized with CTL epitope alone or MHC Class II epitope+CTL epitope. Covalent coupling of Ii-Key/MHC Class II hybrids and CTL epitopes, or MHC Class II sequences within which CTL epitopes are resident, will also provide enhanced CTL responses. In addition, minigenes and DNA vaccines composed of Ii-Key/MHC Class II hybrids CTL sequences will also induce enhanced CTL reactions.
Sequence CWU
1
905116PRTUnknown OrganismDescription of Unknown Organism Mammalian
Ii-key peptide 1Leu Arg Met Lys Leu Pro Lys Pro Pro Lys Pro Val Ser Lys
Met Arg1 5 10
15216PRTArtificial SequenceDescription of Artificial Sequence Synthetic
mammalian Ii-key peptide 2Tyr Arg Met Lys Leu Pro Lys Pro Pro Lys Pro
Val Ser Lys Met Arg1 5 10
1534PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key peptide 3Leu Arg Met Lys147PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key peptide 4Leu Arg Met Lys Leu
Pro Lys1 558PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key peptide 5Leu Arg Met Lys Leu Pro Lys Ser1
5611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key peptide 6Leu Arg Met Lys Leu Pro Lys Ser Ala Lys
Pro1 5 10714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key peptide
7Leu Arg Met Lys Leu Pro Lys Ser Ala Lys Pro Val Ser Lys1 5
1084PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key peptide 8Leu Arg Met Lys195PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key peptide
9Leu Arg Met Lys Xaa1 510626PRTArachis hypogaea 10Met Arg
Gly Arg Val Ser Pro Leu Met Leu Leu Leu Gly Ile Leu Val1 5
10 15Leu Ala Ser Val Ser Ala Thr His
Ala Lys Ser Ser Pro Tyr Gln Lys 20 25
30Lys Thr Glu Asn Pro Cys Ala Gln Arg Cys Leu Gln Ser Cys Gln
Gln 35 40 45Glu Pro Asp Asp Leu
Lys Gln Lys Ala Cys Glu Ser Arg Cys Thr Lys 50 55
60Leu Glu Tyr Asp Pro Arg Cys Val Tyr Asp Pro Arg Gly His
Thr Gly65 70 75 80Thr
Thr Asn Gln Arg Ser Pro Pro Gly Glu Arg Thr Arg Gly Arg Gln
85 90 95Pro Gly Asp Tyr Asp Asp Asp
Arg Arg Gln Pro Arg Arg Glu Glu Gly 100 105
110Gly Arg Trp Gly Pro Ala Gly Pro Arg Glu Arg Glu Arg Glu
Glu Asp 115 120 125Trp Arg Gln Pro
Arg Glu Asp Trp Arg Arg Pro Ser His Gln Gln Pro 130
135 140Arg Lys Ile Arg Pro Glu Gly Arg Glu Gly Glu Gln
Glu Trp Gly Thr145 150 155
160Pro Gly Ser His Val Arg Glu Glu Thr Ser Arg Asn Asn Pro Phe Tyr
165 170 175Phe Pro Ser Arg Arg
Phe Ser Thr Arg Tyr Gly Asn Gln Asn Gly Arg 180
185 190Ile Arg Val Leu Gln Arg Phe Asp Gln Arg Ser Arg
Gln Phe Gln Asn 195 200 205Leu Gln
Asn His Arg Ile Val Gln Ile Glu Ala Lys Pro Asn Thr Leu 210
215 220Val Leu Pro Lys His Ala Asp Ala Asp Asn Ile
Leu Val Ile Gln Gln225 230 235
240Gly Gln Ala Thr Val Thr Val Ala Asn Gly Asn Asn Arg Lys Ser Phe
245 250 255Asn Leu Asp Glu
Gly His Ala Leu Arg Ile Pro Ser Gly Phe Ile Ser 260
265 270Tyr Ile Leu Asn Arg His Asp Asn Gln Asn Leu
Arg Val Ala Lys Ile 275 280 285Ser
Met Pro Val Asn Thr Pro Gly Gln Phe Glu Asp Phe Phe Pro Ala 290
295 300Ser Ser Arg Asp Gln Ser Ser Tyr Leu Gln
Gly Phe Ser Arg Asn Thr305 310 315
320Leu Glu Ala Ala Phe Asn Ala Glu Phe Asn Glu Ile Arg Arg Val
Leu 325 330 335Leu Glu Glu
Asn Ala Gly Gly Glu Gln Glu Glu Arg Gly Gln Arg Arg 340
345 350Trp Ser Thr Arg Ser Ser Glu Asn Asn Glu
Gly Val Ile Val Lys Val 355 360
365Ser Lys Glu His Val Glu Glu Leu Thr Lys His Ala Lys Ser Val Ser 370
375 380Lys Lys Gly Ser Glu Glu Glu Gly
Asp Ile Thr Asn Pro Ile Asn Leu385 390
395 400Arg Glu Gly Glu Pro Asp Leu Ser Asn Asn Phe Gly
Lys Leu Phe Glu 405 410
415Val Lys Pro Asp Lys Lys Asn Pro Gln Leu Gln Asp Leu Asp Met Met
420 425 430Leu Thr Cys Val Glu Ile
Lys Glu Gly Ala Leu Met Leu Pro His Phe 435 440
445Asn Ser Lys Ala Met Val Ile Val Val Val Asn Lys Gly Thr
Gly Asn 450 455 460Leu Glu Leu Val Ala
Val Arg Lys Glu Gln Gln Gln Arg Gly Arg Arg465 470
475 480Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu
Glu Gly Ser Asn Arg Glu 485 490
495Val Arg Arg Tyr Thr Ala Arg Leu Lys Glu Gly Asp Val Phe Ile Met
500 505 510Pro Ala Ala His Pro
Val Ala Ile Asn Ala Ser Ser Glu Leu His Leu 515
520 525Leu Gly Phe Gly Ile Asn Ala Glu Asn Asn His Arg
Ile Phe Leu Ala 530 535 540Gly Asp Lys
Asp Asn Val Ile Asp Gln Ile Glu Lys Gln Ala Lys Asp545
550 555 560Leu Ala Phe Pro Gly Ser Gly
Glu Gln Val Glu Lys Leu Ile Lys Asn 565
570 575Gln Lys Glu Ser His Phe Val Ser Ala Arg Pro Gln
Ser Gln Ser Gln 580 585 590Ser
Pro Ser Ser Pro Glu Lys Glu Ser Pro Glu Lys Glu Asp Gln Glu 595
600 605Glu Glu Asn Gln Gly Gly Lys Gly Pro
Leu Leu Ser Ile Leu Lys Ala 610 615
620Phe Asn625119PRTArachis hypogaea 11Val Lys Pro Asp Lys Lys Asn Pro
Gln1 5129PRTArachis hypogaea 12Ile Arg Val Leu Gln Arg Phe
Asp Gln1 5139PRTArachis hypogaea 13Leu Gln Gly Phe Ser Arg
Asn Thr Leu1 5149PRTArachis hypogaea 14Met Val Ile Val Val
Val Asn Lys Gly1 5159PRTArachis hypogaea 15Val Val Asn Lys
Gly Thr Gly Asn Leu1 5169PRTArachis hypogaea 16Val Arg Arg
Tyr Thr Ala Arg Leu Lys1 5179PRTArachis hypogaea 17Leu Gln
Asn His Arg Ile Val Gln Ile1 5189PRTArachis hypogaea 18Phe
Gln Asn Leu Gln Asn His Arg Ile1 5199PRTArachis hypogaea
19Met Leu Leu Leu Gly Ile Leu Val Leu1 5209PRTArachis
hypogaea 20Leu Leu Gly Ile Leu Val Leu Ala Ser1
5219PRTArachis hypogaea 21Met Arg Gly Arg Val Ser Pro Leu Met1
5229PRTArachis hypogaea 22Leu Val Leu Ala Ser Val Ser Ala Thr1
5239PRTArachis hypogaea 23Leu Asp Met Met Leu Thr Cys Val Glu1
5249PRTArachis hypogaea 24Leu Arg Ile Pro Ser Gly Phe Ile Ser1
5259PRTArachis hypogaea 25Phe Ile Ser Tyr Ile Leu Asn Arg
His1 5269PRTArachis hypogaea 26Leu Asn Arg His Asp Asn Gln
Asn Leu1 5279PRTArachis hypogaea 27Phe Asn Ala Glu Phe Asn
Glu Ile Arg1 5289PRTArachis hypogaea 28Phe Asn Glu Ile Arg
Arg Val Leu Leu1 5299PRTArachis hypogaea 29Val Leu Leu Glu
Glu Asn Ala Gly Gly1 53014PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/Ara h 1 hybrid peptide 30Leu
Arg Met Lys Xaa Ile Arg Val Leu Gln Arg Phe Asp Gln1 5
103114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ara h 1 hybrid peptide 31Leu Arg Met Lys
Xaa Met Arg Gly Arg Val Ser Pro Leu Met1 5
103228PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 1 hybrid peptide 32Leu Arg Met Lys Xaa Met Arg Gly Arg
Val Ser Pro Leu Met Leu Leu1 5 10
15Leu Gly Ile Leu Val Leu Ala Ser Val Ser Ala Thr 20
253314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ara h 1 hybrid peptide 33Leu Arg Met Lys
Xaa Phe Gln Asn Leu Gln Asn His Arg Ile1 5
103417PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 1 hybrid peptide 34Leu Arg Met Lys Xaa Phe Gln Asn Leu
Gln Asn His Arg Ile Val Gln1 5 10
15Ile3514PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ara h 1 hybrid peptide 35Leu Arg Met Lys
Xaa Leu Asp Met Met Leu Thr Cys Val Glu1 5
103614PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 1 hybrid peptide 36Leu Arg Met Lys Xaa Leu Arg Ile Pro
Ser Gly Phe Ile Ser1 5
103725PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 1 hybrid peptide 37Leu Arg Met Lys Xaa Leu Arg Ile Pro
Ser Gly Phe Ile Ser Tyr Ile1 5 10
15Leu Asn Arg His Asp Asn Gln Asn Leu 20
253810PRTArachis hypogaea 38Asn Asn Phe Gly Lys Leu Phe Glu Val Lys1
5 103910PRTArachis hypogaea 39Ser Tyr Leu
Gln Glu Phe Ser Arg Asn Thr1 5
104010PRTArachis hypogaea 40Arg Arg Tyr Thr Ala Arg Leu Lys Glu Gly1
5 104110PRTArachis hypogaea 41Phe Asn Ala Glu
Phe Asn Glu Ile Arg Arg1 5
104210PRTArachis hypogaea 42Gly Thr Gly Asn Leu Glu Leu Val Ala Val1
5 104322PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Ara h 1 hybrid peptide 43Leu
Arg Met Lys Xaa Asn Asn Phe Gly Lys Leu Phe Glu Val Lys Pro1
5 10 15Asp Lys Lys Asn Pro Gln
204414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Ara h 1 hybrid peptide 44Leu Arg Met Lys Xaa Leu
Gln Gly Phe Ser Arg Asn Thr Leu1 5
104514PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 1 hybrid peptide 45Leu Arg Met Lys Xaa Val Arg Arg Tyr
Thr Ala Arg Leu Lys1 5
104614PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 1 hybrid peptide 46Leu Arg Met Lys Xaa Met Val Ile Val
Val Val Asn Lys Gly1 5
104714PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 1 hybrid peptide 47Leu Arg Met Lys Xaa Val Val Asn Lys
Gly Thr Gly Asn Leu1 5
104823PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 1 hybrid peptide 48Leu Arg Met Lys Xaa Met Val Ile Val
Val Val Asn Lys Gly Thr Gly1 5 10
15Asn Leu Glu Leu Val Ala Val 204914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ara h 1
hybrid peptide 49Leu Arg Met Lys Xaa Phe Asn Ala Glu Phe Asn Glu Ile Arg1
5 105014PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/Ara h 1 hybrid peptide 50Leu
Arg Met Lys Xaa Phe Asn Glu Ile Arg Arg Val Leu Leu1 5
105114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ara h 1 hybrid peptide 51Leu Arg Met Lys
Xaa Val Leu Leu Glu Glu Asn Ala Gly Gly1 5
105224PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 1 hybrid peptide 52Leu Arg Met Lys Xaa Phe Asn Ala Glu
Phe Asn Glu Ile Arg Arg Val1 5 10
15Leu Leu Glu Glu Asn Ala Gly Gly 2053156PRTArachis
hypogaea 53Met Ala Lys Leu Thr Ile Leu Val Ala Leu Ala Leu Phe Leu Leu
Ala1 5 10 15Ala His Ala
Ser Ala Arg Gln Gln Trp Glu Leu Gln Gly Asp Arg Arg 20
25 30Cys Gln Ser Gln Leu Glu Arg Ala Asn Leu
Arg Pro Cys Glu Gln His 35 40
45Leu Met Gln Lys Ile Gln Arg Asp Glu Asp Ser Tyr Glu Arg Asp Pro 50
55 60Tyr Ser Pro Ser Gln Asp Pro Tyr Ser
Pro Ser Pro Tyr Asp Arg Arg65 70 75
80Gly Ala Gly Ser Ser Gln His Gln Glu Arg Cys Cys Asn Glu
Leu Asn 85 90 95Glu Phe
Glu Asn Asn Gln Arg Cys Met Cys Glu Ala Leu Gln Gln Ile 100
105 110Met Glu Asn Gln Ser Asp Arg Leu Gln
Gly Arg Gln Gln Glu Gln Gln 115 120
125Phe Lys Arg Glu Leu Arg Asn Leu Pro Gln Gln Cys Gly Leu Arg Ala
130 135 140Pro Gln Arg Cys Asp Leu Asp
Val Glu Ser Gly Gly145 150
1555415PRTArachis hypogaea 54Arg Gln Gln Trp Glu Leu Gln Gly Asp Arg Arg
Cys Gln Ser Gln1 5 10
155520PRTArachis hypogaea 55Leu Arg Pro Cys Glu Gln His Leu Met Gln Lys
Ile Gln Arg Asp Glu1 5 10
15Asp Ser Tyr Glu 205610PRTArachis hypogaea 56His Gln Glu Arg
Cys Cys Asn Glu Leu Asn1 5
105710PRTArachis hypogaea 57Gln Arg Cys Met Cys Glu Ala Leu Gln Gln1
5 105810PRTArachis hypogaea 58Pro Gln Gln Cys
Gly Leu Arg Ala Pro Gln1 5
105920PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 2 hybrid peptide 59Leu Arg Met Lys Xaa Arg Gln Gln Trp
Glu Leu Gln Gly Asp Arg Arg1 5 10
15Cys Gln Ser Gln 206025PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ara h 2
hybrid peptide 60Leu Arg Met Lys Xaa Leu Arg Pro Cys Glu Gln His Leu Met
Gln Lys1 5 10 15Ile Gln
Arg Asp Glu Asp Ser Tyr Glu 20
256115PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 2 hybrid peptide 61Leu Arg Met Lys Xaa His Gln Glu Arg
Cys Cys Asn Glu Leu Asn1 5 10
156215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Ara h 2 hybrid peptide 62Leu Arg Met Lys Xaa Gln
Arg Cys Met Cys Glu Ala Leu Gln Gln1 5 10
156315PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ara h 2 hybrid peptide 63Leu Arg Met Lys
Xaa Pro Gln Gln Cys Gly Leu Arg Ala Pro Gln1 5
10 15649PRTArachis hypogaea 64Ile Leu Val Ala Leu
Ala Leu Phe Leu1 5659PRTArachis hypogaea 65Leu Gln Gly Asp
Arg Arg Cys Gln Ser1 5669PRTArachis hypogaea 66Leu Thr Ile
Leu Val Ala Leu Ala Leu1 5679PRTArachis hypogaea 67Leu Met
Gln Lys Ile Gln Arg Asp Glu1 5689PRTArachis hypogaea 68Leu
Phe Leu Leu Ala Ala His Ala Ser1 5699PRTArachis hypogaea
69Leu Val Ala Leu Ala Leu Phe Leu Leu1 5709PRTArachis
hypogaea 70Leu Arg Pro Cys Glu Gln His Leu Met1
5719PRTArachis hypogaea 71Leu Ala Leu Phe Leu Leu Ala Ala His1
5729PRTArachis hypogaea 72Leu Arg Asn Leu Pro Gln Gln Cys Gly1
5739PRTArachis hypogaea 73Leu Glu Arg Ala Asn Leu Arg Pro Cys1
5749PRTArachis hypogaea 74Phe Leu Leu Ala Ala His Ala Ser Ala1
5759PRTArachis hypogaea 75Tyr Asp Arg Arg Gly Ala Gly Ser
Ser1 5769PRTArachis hypogaea 76Phe Glu Asn Asn Gln Arg Cys
Met Cys1 57714PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Ara h 2 hybrid peptide 77Leu
Arg Met Lys Xaa Ile Leu Val Ala Leu Ala Leu Phe Leu1 5
107814PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ara h 2 hybrid peptide 78Leu Arg Met Lys
Xaa Leu Thr Ile Leu Val Ala Leu Ala Leu1 5
107914PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 2 hybrid peptide 79Leu Arg Met Lys Xaa Leu Val Ala Leu
Ala Leu Phe Leu Leu1 5
108017PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 2 hybrid peptide 80Leu Arg Met Lys Xaa Leu Thr Ile Leu
Val Ala Leu Ala Leu Phe Leu1 5 10
15Leu8114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ara h 2 hybrid peptide 81Leu Arg Met Lys
Xaa Leu Arg Asn Leu Pro Gln Gln Cys Gly1 5
108214PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 2 hybrid peptide 82Leu Arg Met Lys Xaa Tyr Asp Arg Arg
Gly Ala Gly Ser Ser1 5
108314PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 2 hybrid peptide 83Leu Arg Met Lys Xaa Phe Glu Asn Asn
Gln Arg Cys Met Cys1 5 108410PRTArachis
hypogaea 84His Ala Ser Ala Arg Gln Gln Trp Glu Leu1 5
108510PRTArachis hypogaea 85Gln Trp Glu Leu Gln Gly Asp Arg
Arg Cys1 5 108610PRTArachis hypogaea
86Asp Arg Arg Cys Gln Ser Gln Leu Glu Arg1 5
108710PRTArachis hypogaea 87Leu Arg Pro Cys Glu Gln His Leu Met Gln1
5 108810PRTArachis hypogaea 88Lys Ile Gln
Arg Asp Glu Asp Ser Tyr Glu1 5
108910PRTArachis hypogaea 89Lys Arg Glu Leu Arg Asn Leu Pro Gln Gln1
5 109014PRTArtificial SequenceDescription of
Artificial Sequence Synthetic hybrid peptide containing predicted
MHC Class II Ara h 2 epitope 90Leu Arg Met Lys Xaa Leu Gln Gly Asp
Arg Arg Cys Gln Ser1 5
109114PRTArtificial SequenceDescription of Artificial Sequence Synthetic
hybrid peptide containing predicted MHC Class II Ara h 2 epitope
91Leu Arg Met Lys Xaa Leu Met Gln Lys Ile Gln Arg Asp Glu1
5 109214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic hybrid peptide containing predicted MHC Class
II Ara h 2 epitope 92Leu Arg Met Lys Xaa Leu Arg Pro Cys Glu Gln His Leu
Met1 5 109320PRTArtificial
SequenceDescription of Artificial Sequence Synthetic hybrid peptide
containing predicted MHC Class II Ara h 2 epitope 93Leu Arg Met Lys
Xaa Leu Arg Pro Cys Glu Gln His Leu Met Lys Ile1 5
10 15Gln Arg Asp Glu
209420PRTArtificial SequenceDescription of Artificial Sequence Synthetic
hybrid peptide containing predicted MHC Class II Ara h 2 epitope
94Leu Arg Met Lys Xaa Leu Glu Arg Ala Asn Leu Arg Pro Cys Glu Gln1
5 10 15His Leu Met
Gln209520PRTArtificial SequenceDescription of Artificial Sequence
Synthetic hybrid peptide containing predicted MHC Class II Ara
h 2 epitope 95Leu Arg Met Lys Xaa Phe Leu Leu Ala Ala His Ala Ser Ala Arg
Gln1 5 10 15Gln Trp Glu
Leu 2096507PRTArachis hypogaea 96Arg Gln Gln Pro Glu Glu Asn
Ala Cys Gln Phe Gln Arg Leu Asn Ala1 5 10
15Gln Arg Pro Asp Asn Arg Ile Glu Ser Glu Gly Gly Tyr
Ile Glu Thr 20 25 30Trp Asn
Pro Asn Asn Gln Glu Phe Glu Cys Ala Gly Val Ala Leu Ser 35
40 45Arg Leu Val Leu Arg Arg Asn Ala Leu Arg
Arg Pro Phe Tyr Ser Asn 50 55 60Ala
Pro Gln Glu Ile Phe Ile Gln Gln Gly Arg Gly Tyr Phe Gly Leu65
70 75 80Ile Phe Pro Gly Cys Pro
Arg His Tyr Glu Glu Pro His Thr Gln Gly 85
90 95Arg Arg Ser Gln Ser Gln Arg Pro Pro Arg Arg Leu
Gln Gly Glu Asp 100 105 110Gln
Ser Gln Gln Gln Arg Asp Ser His Gln Lys Val His Arg Phe Asp 115
120 125Glu Gly Asp Leu Ile Ala Val Pro Thr
Gly Val Ala Phe Trp Leu Tyr 130 135
140Asn Asp His Asp Thr Asp Val Val Ala Val Ser Leu Thr Asp Thr Asn145
150 155 160Asn Asn Asp Asn
Gln Leu Asp Gln Phe Pro Arg Arg Phe Asn Leu Ala 165
170 175Gly Asn Thr Glu Gln Glu Phe Leu Arg Tyr
Gln Gln Gln Ser Arg Gln 180 185
190Ser Arg Arg Arg Ser Leu Pro Tyr Ser Pro Tyr Ser Pro Gln Ser Gln
195 200 205Pro Arg Gln Glu Glu Arg Glu
Phe Ser Pro Arg Gly Gln His Ser Arg 210 215
220Arg Glu Arg Ala Gly Gln Glu Glu Glu Asn Glu Gly Gly Asn Ile
Phe225 230 235 240Ser Gly
Phe Thr Pro Glu Phe Leu Glu Gln Ala Phe Gln Val Asp Asp
245 250 255Arg Gln Ile Val Gln Asn Leu
Arg Gly Glu Thr Glu Ser Glu Glu Glu 260 265
270Gly Ala Ile Val Thr Val Arg Gly Gly Leu Arg Ile Leu Ser
Pro Asp 275 280 285Arg Lys Arg Arg
Ala Asp Glu Glu Glu Glu Tyr Asp Glu Asp Glu Tyr 290
295 300Glu Tyr Asp Glu Glu Asp Arg Arg Arg Gly Arg Gly
Ser Arg Gly Arg305 310 315
320Gly Asn Gly Ile Glu Glu Thr Ile Cys Thr Ala Ser Ala Lys Lys Asn
325 330 335Ile Gly Arg Asn Arg
Ser Pro Asp Ile Tyr Asn Pro Gln Ala Gly Ser 340
345 350Leu Lys Thr Ala Asn Asp Leu Asn Leu Leu Ile Leu
Arg Trp Leu Gly 355 360 365Pro Ser
Ala Glu Tyr Gly Asn Leu Tyr Arg Asn Ala Leu Phe Val Ala 370
375 380His Tyr Asn Thr Asn Ala His Ser Ile Ile Tyr
Arg Leu Arg Gly Arg385 390 395
400Ala His Val Gln Val Val Asp Ser Asn Gly Asn Arg Val Tyr Asp Glu
405 410 415Glu Leu Gln Glu
Gly His Val Leu Val Val Pro Gln Asn Phe Ala Val 420
425 430Ala Gly Lys Ser Gln Ser Glu Asn Phe Glu Tyr
Val Ala Phe Lys Thr 435 440 445Asp
Ser Arg Pro Ser Ile Ala Asn Leu Ala Gly Glu Asn Ser Val Ile 450
455 460Asp Asn Leu Pro Glu Glu Val Val Ala Asn
Ser Tyr Gly Leu Gln Arg465 470 475
480Glu Gln Ala Arg Gln Leu Lys Asn Asn Asn Pro Phe Lys Phe Phe
Val 485 490 495Pro Pro Ser
Gln Gln Ser Pro Arg Ala Val Ala 500
505979PRTArachis hypogaea 97Tyr Arg Leu Arg Gly Arg Ala His Val1
5989PRTArachis hypogaea 98Ile Ile Tyr Arg Leu Arg Gly Arg Ala1
5999PRTArachis hypogaea 99Phe Lys Thr Asp Ser Arg Pro Ser Ile1
51009PRTArachis hypogaea 100Val Arg Gly Gly Leu Arg Ile Leu
Ser1 51019PRTArachis hypogaea 101Ile Val Thr Val Arg Gly
Gly Leu Arg1 51029PRTArachis hypogaea 102Leu Arg Ile Leu
Ser Pro Asp Arg Lys1 51039PRTArachis hypogaea 103Phe Gln
Val Asp Asp Arg Gln Ile Val1 51049PRTArachis hypogaea
104Leu Arg Trp Leu Gly Pro Ser Ala Glu1 51059PRTArachis
hypogaea 105Leu Ile Leu Arg Trp Leu Gly Pro Ser1
51069PRTArachis hypogaea 106Phe Asn Leu Ala Gly Asn Thr Glu Gln1
51079PRTArachis hypogaea 107Leu Val Val Pro Gln Asn Phe Ala Val1
51089PRTArachis hypogaea 108Val Gln Val Val Asp Ser Asn Gly
Asn1 51099PRTArachis hypogaea 109Val Val Asp Ser Asn Gly
Asn Arg Val1 51109PRTArachis hypogaea 110Phe Val Ala His
Tyr Asn Thr Asn Ala1 511114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ara h 3
hybrid peptide 111Leu Arg Met Lys Xaa Ile Ile Tyr Arg Leu Arg Gly Arg
Ala1 5 1011214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ara h 3
hybrid peptide 112Leu Arg Met Lys Xaa Tyr Arg Leu Arg Gly Arg Ala His
Val1 5 1011316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ara h 3
hybrid peptide 113Leu Arg Met Lys Xaa Ile Ile Tyr Arg Leu Arg Gly Arg Ala
His Val1 5 10
1511414PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 3 hybrid peptide 114Leu Arg Met Lys Xaa Phe Lys Thr Asp
Ser Arg Pro Ser Ile1 5
1011514PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 3 hybrid peptide 115Leu Arg Met Lys Xaa Leu Ile Leu Arg
Trp Leu Gly Pro Ser1 5
1011614PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 3 hybrid peptide 116Leu Arg Met Lys Xaa Leu Arg Trp Leu
Gly Pro Ser Ala Glu1 5
1011716PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 3 hybrid peptide 117Leu Arg Met Lys Xaa Leu Ile Leu Arg
Trp Leu Gly Pro Ser Ala Glu1 5 10
1511814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Ara h 3 hybrid peptide 118Leu Arg Met Lys Xaa Phe
Gln Val Asp Asp Arg Gln Ile Val1 5
1011914PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 3 hybrid peptide 119Leu Arg Met Lys Xaa Phe Asn Leu Ala
Gly Asn Thr Glu Gln1 5
1012014PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 3 hybrid peptide 120Leu Arg Met Lys Xaa Leu Val Val Pro
Gln Asn Phe Ala Val1 5
1012114PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 3 hybrid peptide 121Leu Arg Met Lys Xaa Val Gln Val Val
Asp Ser Asn Gly Asn1 5
1012214PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 3 hybrid peptide 122Leu Arg Met Lys Xaa Val Val Asp Ser
Asn Gly Asn Arg Val1 5
1012316PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ara h 3 hybrid peptide 123Leu Arg Met Lys Xaa Val Gln Val Val
Asp Ser Asn Gly Asn Arg Val1 5 10
1512414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Ara h 3 hybrid peptide 124Leu Arg Met Lys Xaa Phe
Val Ala His Tyr Asn Thr Asn Ala1 5
1012515PRTArachis hypogaea 125Val Thr Val Arg Gly Gly Leu Arg Ile Leu Ser
Pro Asp Arg Lys1 5 10
1512614PRTArtificial SequenceDescription of Artificial Sequence Synthetic
hybrid peptide containing predicted Class II Ara h 3 epitope
126Leu Arg Met Lys Xaa Ile Val Thr Val Arg Gly Gly Leu Arg1
5 1012714PRTArtificial SequenceDescription of
Artificial Sequence Synthetic hybrid peptide containing predicted
Class II Ara h 3 epitope 127Leu Arg Met Lys Xaa Val Arg Gly Gly Leu
Arg Ile Leu Ser1 5 1012821PRTArtificial
SequenceDescription of Artificial Sequence Synthetic hybrid peptide
containing predicted Class II Ara h 3 epitope 128Leu Arg Met Lys Xaa
Ile Val Thr Val Arg Gly Gly Leu Arg Ile Leu1 5
10 15Ser Pro Asp Arg Lys
2012921PRTArtificial SequenceDescription of Artificial Sequence Synthetic
hybrid peptide containing predicted Class II Ara h 3 epitope
129Leu Arg Met Lys Xaa Ile Val Thr Val Arg Gly Gly Leu Arg Ile Leu1
5 10 15Ser Pro Asp Arg Lys
2013017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Th1-skewing peptide 130Ile Ser Gln Ala Val His Ala Ala His
Ala Glu Ile Asn Ala Ala Gly1 5 10
15Arg13117PRTUnknown OrganismDescription of Unknown Organism
Wild type peptide 131Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile
Asn Glu Ala Gly1 5 10
15Arg13217PRTFelis domesticus 132Leu Phe Leu Thr Gly Thr Pro Asp Glu Tyr
Val Glu Gln Val Ala Gln1 5 10
15Tyr13316PRTFelis domesticus 133Glu Gln Val Ala Gln Tyr Lys Ala Leu
Pro Val Val Leu Glu Asn Ala1 5 10
1513417PRTFelis domesticus 134Lys Ala Leu Pro Val Val Leu Glu
Asn Ala Arg Ile Leu Lys Asn Cys1 5 10
15Val13588PRTFelis domesticus 135Met Leu Asp Ala Ala Leu Pro
Pro Cys Pro Thr Val Ala Ala Thr Ala1 5 10
15Asp Cys Glu Ile Cys Pro Ala Val Lys Arg Asp Val Asp
Leu Phe Leu 20 25 30Thr Gly
Thr Pro Asp Glu Tyr Val Glu Gln Val Ala Gln Tyr Lys Ala 35
40 45Leu Pro Val Val Leu Glu Asn Ala Arg Ile
Leu Lys Asn Cys Val Asp 50 55 60Ala
Lys Met Thr Glu Glu Asp Lys Glu Asn Ala Leu Ser Leu Leu Asp65
70 75 80Lys Ile Tyr Thr Ser Pro
Leu Cys 85136109PRTFelis domesticus 136Met Arg Gly Ala Leu
Leu Val Leu Ala Leu Leu Val Thr Gln Ala Leu1 5
10 15Gly Val Lys Met Ala Glu Thr Cys Pro Ile Phe
Tyr Asp Val Phe Phe 20 25
30Ala Val Ala Asn Gly Asn Glu Leu Leu Leu Asp Leu Ser Leu Thr Lys
35 40 45Val Asn Ala Thr Glu Pro Glu Arg
Thr Ala Met Lys Lys Ile Gln Asp 50 55
60Cys Tyr Val Glu Asn Gly Leu Ile Ser Arg Val Leu Asp Gly Leu Val65
70 75 80Met Thr Thr Ile Ser
Ser Ser Lys Asp Cys Met Gly Glu Ala Val Gln 85
90 95Asn Thr Val Glu Asp Leu Lys Leu Asn Thr Leu
Gly Arg 100 1051379PRTFelis domesticus 137Tyr
Lys Ala Leu Pro Val Val Leu Glu1 51389PRTFelis domesticus
138Val Val Leu Glu Asn Ala Arg Ile Leu1 51399PRTFelis
domesticus 139Val Ala Gln Tyr Lys Ala Leu Pro Val1
51409PRTFelis domesticus 140Tyr Val Glu Gln Val Ala Gln Tyr Lys1
51419PRTFelis domesticus 141Val Lys Arg Asp Val Asp Leu Phe Leu1
51429PRTFelis domesticus 142Ile Cys Pro Ala Val Lys Arg Asp
Val1 51439PRTFelis domesticus 143Leu Ser Leu Leu Asp Lys
Ile Tyr Thr1 51449PRTFelis domesticus 144Leu Asp Lys Ile
Tyr Thr Ser Pro Leu1 51459PRTFelis domesticus 145Leu Phe
Leu Thr Gly Thr Pro Asp Glu1 514614PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Fel d 1
(chain 1) hybrid peptide 146Leu Arg Met Lys Xaa Val Ala Gln Tyr Lys Ala
Leu Pro Val1 5 1014714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Fel d 1
(chain 1) hybrid peptide 147Leu Arg Met Lys Xaa Tyr Lys Ala Leu Pro Val
Val Leu Glu1 5 1014814PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Fel d 1
(chain 1) hybrid peptide 148Leu Arg Met Lys Xaa Val Val Leu Glu Asn Ala
Arg Ile Leu1 5 1014914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Fel d 1
(chain 1) hybrid peptide 149Leu Arg Met Lys Xaa Tyr Val Glu Gln Val Ala
Gln Tyr Lys1 5 1015026PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Fel d 1
(chain 1) hybrid peptide 150Leu Arg Met Lys Xaa Tyr Val Glu Gln Val Ala
Gln Tyr Lys Ala Leu1 5 10
15Pro Val Val Leu Glu Asn Ala Arg Ile Leu 20
2515114PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 151Leu Arg Met Lys Xaa Ile
Cys Pro Ala Val Lys Arg Asp Val1 5
1015214PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 152Leu Arg Met Lys Xaa Val
Lys Arg Asp Val Asp Leu Phe Leu1 5
1015314PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 153Leu Arg Met Lys Xaa Leu
Phe Leu Thr Gly Thr Pro Asp Glu1 5
1015424PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 154Leu Arg Met Lys Xaa Ile
Cys Pro Ala Val Lys Arg Asp Val Asp Leu1 5
10 15Phe Leu Thr Gly Thr Pro Asp Glu
2015514PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 155Leu Arg Met Lys Xaa Leu
Ser Leu Leu Asp Lys Ile Tyr Thr1 5
1015614PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 156Leu Arg Met Lys Xaa Leu
Asp Lys Ile Tyr Thr Ser Pro Leu1 5
1015717PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 157Leu Arg Met Lys Xaa Leu
Ser Leu Leu Asp Lys Ile Tyr Thr Ser Pro1 5
10 15Leu15817PRTFelis domesticus 158Glu Ile Cys Pro Ala
Val Lys Arg Asp Val Asp Leu Phe Leu Thr Gly1 5
10 15Thr15917PRTFelis domesticus 159Leu Phe Leu Thr
Gly Thr Pro Asp Glu Tyr Val Glu Gln Val Ala Gln1 5
10 15Tyr16016PRTFelis domesticus 160Glu Gln Val
Ala Gln Tyr Lys Ala Leu Pro Val Val Leu Glu Asn Ala1 5
10 1516117PRTFelis domesticus 161Lys Ala
Leu Pro Val Val Leu Glu Asn Ala Arg Ile Leu Lys Asn Cys1 5
10 15Val16217PRTFelis domesticus 162Arg
Ile Leu Lys Asn Cys Val Asp Ala Lys Met Thr Glu Glu Asp Lys1
5 10 15Glu16316PRTFelis domesticus
163Lys Met Thr Glu Glu Asp Lys Glu Asn Ala Leu Ser Leu Leu Asp Lys1
5 10 1516416PRTFelis
domesticus 164Lys Glu Asn Ala Leu Ser Val Leu Asp Lys Ile Tyr Thr Ser Pro
Leu1 5 10
1516522PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 165Leu Arg Met Lys Xaa Leu
Phe Leu Thr Gly Thr Pro Asp Glu Tyr Val1 5
10 15Glu Gln Val Ala Gln Tyr
2016621PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 166Leu Arg Met Lys Xaa Glu
Gln Val Ala Gln Tyr Lys Ala Leu Pro Val1 5
10 15Val Leu Glu Asn Ala
2016722PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 167Leu Arg Met Lys Xaa Lys
Ala Leu Pro Val Val Leu Glu Asn Ala Arg1 5
10 15Ile Leu Lys Asn Cys Val
2016822PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 168Leu Arg Met Lys Xaa Arg
Ile Leu Lys Asn Cys Val Asp Ala Lys Met1 5
10 15Thr Glu Glu Asp Lys Glu
2016937PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 1) hybrid peptide 169Leu Arg Met Lys Xaa Gln
Val Ala Gln Tyr Lys Ala Leu Pro Val Val1 5
10 15Leu Glu Asn Ala Arg Ile Leu Lys Asn Cys Val Asp
Ala Lys Met Thr 20 25 30Glu
Glu Asp Lys Glu 351709PRTFelis domesticus 170Leu Leu Val Thr Gln
Ala Leu Gly Val1 51719PRTFelis domesticus 171Leu Leu Leu
Asp Leu Ser Leu Thr Lys1 51729PRTFelis domesticus 172Leu
Val Met Thr Thr Ile Ser Ser Ser1 51739PRTFelis domesticus
173Val Lys Met Ala Glu Thr Cys Pro Ile1 51749PRTFelis
domesticus 174Leu Val Leu Ala Leu Leu Val Thr Gln1
51759PRTFelis domesticus 175Leu Leu Val Leu Ala Leu Leu Val Thr1
517614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Fel d (chain 2) hybrid peptide 176Leu Arg Met Lys
Xaa Leu Leu Val Thr Gln Ala Leu Gly Val1 5
1017714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Fel d (chain 2) hybrid peptide 177Leu Arg Met Lys
Xaa Leu Val Leu Ala Leu Leu Val Thr Gln1 5
1017814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Fel d (chain 2) hybrid peptide 178Leu Arg Met Lys
Xaa Leu Leu Val Leu Ala Leu Leu Val Thr1 5
1017919PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Fel d (chain 2) hybrid peptide 179Leu Arg Met Lys
Xaa Leu Leu Val Leu Ala Leu Leu Val Thr Gln Ala1 5
10 15Leu Gly Val18014PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Fel d
(chain 2) hybrid peptide 180Leu Arg Met Lys Xaa Val Lys Met Ala Glu Thr
Cys Pro Ile1 5 1018114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Fel d
(chain 2) hybrid peptide 181Leu Arg Met Lys Xaa Leu Leu Leu Asp Leu Ser
Leu Thr Lys1 5 1018214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Fel d
(chain 2) hybrid peptide 182Leu Arg Met Lys Xaa Leu Val Met Thr Thr Ile
Ser Ser Ser1 5 1018316PRTFelis domesticus
183Leu Thr Lys Val Asn Ala Thr Glu Pro Glu Arg Thr Ala Met Lys Lys1
5 10 1518416PRTFelis
domesticus 184Thr Ala Met Lys Lys Ile Gln Asp Cys Tyr Val Glu Asn Gly Leu
Ile1 5 10
1518516PRTFelis domesticus 185Cys Tyr Val Glu Asn Gly Leu Ile Ser Arg Val
Leu Asp Gly Leu Val1 5 10
1518616PRTFelis domesticus 186Ile Ser Ser Ser Lys Asp Cys Met Gly Glu
Ala Val Gln Asn Thr Val1 5 10
1518716PRTFelis domesticus 187Ala Val Gln Asn Thr Val Glu Asp Leu
Lys Leu Asn Thr Leu Gly Arg1 5 10
1518821PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Fel d 1 (chain 2) hybrid peptide 188Leu Arg Met Lys
Xaa Leu Thr Lys Val Asn Ala Thr Glu Pro Glu Arg1 5
10 15Thr Ala Met Lys Lys
2018921PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 2) hybrid peptide 189Leu Arg Met Lys Xaa Thr
Ala Met Lys Lys Ile Gln Asp Cys Tyr Val1 5
10 15Glu Asn Gly Leu Ile
2019021PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 2) hybrid peptide 190Leu Arg Met Lys Xaa Cys
Tyr Val Glu Asn Gly Leu Ile Ser Arg Val1 5
10 15Leu Asp Gly Leu Val
2019121PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 2) hybrid peptide 191Leu Arg Met Lys Xaa Ile
Ser Ser Ser Lys Asp Cys Met Gly Glu Ala1 5
10 15Val Gln Asn Thr Val
2019229PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Fel d 1 (chain 2) hybrid peptide 192Leu Arg Met Lys Xaa Thr
Ala Met Lys Lys Ile Gln Asp Cys Tyr Val1 5
10 15Glu Asn Gly Leu Ile Ser Arg Val Leu Asp Gly Leu
Val 20 25193263PRTPheleum pratense 193Met Ala
Ser Ser Ser Ser Val Leu Leu Val Val Val Leu Phe Ala Val1 5
10 15Phe Leu Gly Ser Ala Tyr Gly Ile
Pro Lys Val Pro Pro Gly Pro Asn 20 25
30Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys Ser Thr
Trp 35 40 45Tyr Gly Lys Pro Thr
Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys 50 55
60Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe Ser Gly Met Thr
Gly Cys65 70 75 80Gly
Asn Thr Pro Ile Phe Lys Ser Gly Arg Gly Cys Gly Ser Cys Phe
85 90 95Glu Ile Lys Cys Thr Lys Pro
Glu Ala Cys Ser Gly Glu Pro Val Val 100 105
110Val His Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Pro Tyr
His Phe 115 120 125Asp Leu Ser Gly
His Ala Phe Gly Ala Met Ala Lys Lys Gly Asp Glu 130
135 140Gln Lys Leu Arg Ser Ala Gly Glu Leu Glu Leu Gln
Phe Arg Arg Val145 150 155
160Lys Cys Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe His Val Glu Lys
165 170 175Gly Ser Asn Pro Asn
Tyr Leu Ala Leu Leu Val Lys Tyr Val Asn Gly 180
185 190Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys
Gly Lys Asp Lys 195 200 205Trp Ile
Glu Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr 210
215 220Pro Asp Lys Leu Thr Gly Pro Phe Thr Val Arg
Tyr Thr Thr Glu Gly225 230 235
240Gly Thr Lys Thr Glu Ala Glu Asp Val Ile Pro Glu Gly Trp Lys Ala
245 250 255Asp Thr Ser Tyr
Glu Ser Lys 2601949PRTPheleum pratense 194Met Ala Ser Ser Ser
Ser Val Leu Leu1 51959PRTPheleum pratense 195Trp Arg Ile
Asp Thr Pro Asp Lys Leu1 51969PRTPheleum pratense 196Val
Val Val Leu Phe Ala Val Phe Leu1 51978PRTPheleum pratense
197Val Val Leu Phe Ala Val Leu Gly1 51989PRTPheleum
pratense 198Val Leu Leu Val Val Val Leu Phe Ala1
51999PRTPheleum pratense 199Phe Glu Ile Lys Cys Thr Lys Pro Glu1
52009PRTPheleum pratense 200Val Phe Leu Gly Ser Ala Tyr Gly Ile1
52019PRTPheleum pratense 201Leu Val Lys Tyr Val Asn Gly Asp
Gly1 52029PRTPheleum pratense 202Leu Leu Val Lys Tyr Val
Asn Gly Asp1 520314PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Phl p 1 hybrid peptide 203Leu
Arg Met Lys Xaa Met Ala Ser Ser Ser Ser Val Leu Leu1 5
1020414PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Phl p 1 hybrid peptide 204Leu Arg Met Lys
Xaa Val Leu Leu Val Val Val Leu Phe Ala1 5
1020514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Phl p 1 hybrid peptide 205Leu Arg Met Lys Xaa Val
Val Val Leu Phe Ala Val Phe Leu1 5
1020614PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Phl p 1 hybrid peptide 206Leu Arg Met Lys Xaa Val Val Leu Phe
Ala Val Phe Leu Gly1 5
1020724PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Phl p 1 hybrid peptide 207Leu Arg Met Lys Xaa Met Ala Ser Ser
Ser Ser Val Leu Leu Val Val1 5 10
15Val Leu Phe Ala Val Phe Leu Gly
2020814PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Phl p 1 hybrid peptide 208Leu Arg Met Lys Xaa Trp Arg Ile Asp
Thr Pro Asp Lys Leu1 5
1020914PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Phl p 1 hybrid peptide 209Leu Arg Met Lys Xaa Phe Glu Ile Lys
Cys Thr Lys Pro Glu1 5
1021014PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Phl p 1 hybrid peptide 210Leu Arg Met Lys Xaa Val Phe Leu Gly
Ser Ala Tyr Gly Ile1 5
1021114PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Phl p 1 hybrid peptide 211Leu Arg Met Lys Xaa Leu Leu Val Lys
Tyr Val Asn Gly Asp1 5
1021214PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Phl p 1 hybrid peptide 212Leu Arg Met Lys Xaa Leu Val Lys Tyr
Val Asn Gly Asp Gly1 5
1021315PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Phl p 1 hybrid peptide 213Leu Arg Met Lys Xaa Leu Leu Val Lys
Tyr Val Asn Gly Asp Gly1 5 10
1521412PRTPheleum pratense 214Phe Glu Ile Lys Cys Thr Lys Pro Glu
Ala Cys Ser1 5 1021512PRTPheleum pratense
215Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala1 5
1021617PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Phl p 1 hybrid peptide 216Leu Arg Met Lys
Xaa Phe Glu Ile Lys Cys Thr Lys Pro Glu Ala Cys1 5
10 15Ser21717PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/Phl p 1 hybrid peptide
217Leu Arg Met Lys Xaa Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His1
5 10 15Ala21830PRTPheleum
pratense 218Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly
Asp1 5 10 15Lys Trp Leu
Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr20 25
3021927PRTPheleum pratense 219Gly Tyr Lys Asp Val Asp Lys Pro
Pro Phe Ser Gly Met Thr Gly Cys1 5 10
15Gly Asn Thr Pro Ile Phe Lys Ser Gly Arg Gly 20
2522031PRTPheleum pratense 220Glu Pro Val Val Val His Ile
Thr Asp Asp Asn Glu Glu Pro Ile Ala1 5 10
15Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ala
Met Ala 20 25
3022127PRTPheleum pratense 221His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu
Ala Leu Leu Val Lys1 5 10
15Tyr Val Asn Gly Asp Gly Asp Val Val Ala Val 20
2522229PRTPheleum pratense 222Arg Tyr Thr Thr Glu Gly Gly Thr Lys Thr
Glu Ala Glu Asp Val Ile1 5 10
15Pro Glu Gly Trp Lys Ala Asp Thr Ser Tyr Glu Ser Lys 20
2522349PRTArtificial SequenceDescription of Artificial
Sequence Synthetic hybrid peptide including an experimentally
defined MHC Class II and IgE binding Php 1 epitope 223Leu Arg Met
Lys Xaa Phe Glu Ile Lys Cys Thr Lys Pro Glu Ala Cys1 5
10 15Ser Gly Glu Pro Val Val Val His Ile
Thr Asp Asp Asn Glu Glu Pro 20 25
30Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ala Met
35 40 45Ala224286PRTPheleum
pratense 224Ala Asp Leu Gly Tyr Gly Pro Ala Thr Pro Ala Ala Pro Ala Ala
Gly1 5 10 15Tyr Thr Pro
Ala Thr Pro Ala Ala Pro Ala Gly Ala Asp Ala Ala Gly 20
25 30Lys Ala Thr Thr Glu Glu Gln Lys Leu Ile
Glu Lys Ile Asn Ala Gly 35 40
45Phe Lys Ala Ala Leu Ala Gly Ala Gly Val Gln Pro Ala Asp Lys Tyr 50
55 60Arg Thr Phe Val Ala Thr Phe Gly Pro
Ala Ser Asn Lys Ala Phe Ala65 70 75
80Glu Gly Leu Ser Gly Glu Pro Lys Gly Ala Ala Glu Ser Ser
Ser Lys 85 90 95Ala Ala
Leu Thr Ser Lys Leu Asp Ala Ala Tyr Lys Leu Ala Tyr Lys 100
105 110Thr Ala Glu Gly Ala Thr Pro Glu Ala
Lys Tyr Asp Ala Tyr Val Ala 115 120
125Thr Leu Ser Glu Ala Leu Arg Ile Ile Ala Gly Thr Leu Glu Val His
130 135 140Ala Val Lys Pro Ala Ala Glu
Glu Val Lys Val Ile Pro Ala Gly Glu145 150
155 160Leu Gln Val Ile Glu Lys Val Asp Ala Ala Phe Lys
Val Ala Ala Thr 165 170
175Ala Ala Asn Ala Ala Pro Ala Asn Asp Lys Phe Thr Val Phe Glu Ala
180 185 190Ala Phe Asn Asp Glu Ile
Lys Ala Ser Thr Gly Gly Ala Tyr Glu Ser 195 200
205Tyr Lys Phe Ile Pro Ala Leu Glu Ala Ala Val Lys Gln Ala
Tyr Ala 210 215 220Ala Thr Val Ala Thr
Ala Pro Glu Val Lys Tyr Thr Val Phe Glu Thr225 230
235 240Ala Leu Lys Lys Ala Ile Thr Ala Met Ser
Glu Ala Gln Lys Ala Ala 245 250
255Lys Pro Ala Ala Ala Ala Thr Ala Thr Ala Thr Ala Ala Val Gly Ala
260 265 270Ala Thr Gly Ala Ala
Thr Ala Ala Thr Gly Gly Tyr Lys Val 275 280
2852259PRTPheleum pratense 225Tyr Val Ala Thr Leu Ser Glu Ala
Leu1 52269PRTPheleum pratense 226Val Lys Val Ile Pro Ala
Gly Glu Leu1 52279PRTPheleum pratense 227Leu Arg Ile Ile
Ala Gly Thr Leu Glu1 52289PRTPheleum pratense 228Tyr Lys
Phe Ile Pro Ala Leu Glu Ala1 52299PRTPheleum pratense
229Tyr Glu Ser Tyr Lys Phe Ile Pro Ala1 52309PRTPheleum
pratense 230Phe Lys Val Ala Ala Thr Ala Ala Asn1
52319PRTPheleum pratense 231Tyr Arg Thr Phe Val Ala Thr Phe Gly1
523212PRTPheleum pratense 232Tyr Lys Phe Ile Pro Ala Leu Glu Ala Ala
Val Lys1 5 1023312PRTPheleum pratense
233Leu Gln Val Ile Glu Lys Val Asp Ala Ala Phe Lys1 5
1023412PRTPheleum pratense 234Tyr Lys Cys Ile Pro Ser Leu Glu
Ala Ala Val Lys1 5 1023512PRTPheleum
pratense 235Leu Gln Ile Ile Asp Lys Ile Asp Ala Ala Phe Lys1
5 1023614PRTArtificial SequenceDescription of
Artificial Sequence Synthetic hybrid containing Phl p 5 MHC Class II
non-overlapping epitope 236Leu Arg Met Lys Xaa Tyr Val Ala Thr Leu
Ser Glu Ala Leu1 5 1023714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic hybrid
containing Phl p 5 MHC Class II non-overlapping epitope 237Leu Arg
Met Lys Xaa Val Lys Val Ile Pro Ala Gly Glu Leu1 5
1023814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic hybrid containing Phl p 5 MHC Class II
non-overlapping epitope 238Leu Arg Met Lys Xaa Leu Arg Ile Ile Ala Gly
Thr Leu Glu1 5 1023914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic hybrid
containing Phl p 5 MHC Class II non-overlapping epitope 239Leu Arg
Met Lys Xaa Tyr Arg Thr Phe Val Ala Thr Phe Gly1 5
1024014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic hybrid containing Phl p 5 MHC Class II overlapping
epitope 240Leu Arg Met Lys Xaa Tyr Lys Phe Ile Pro Ala Leu Glu Ala1
5 1024114PRTArtificial SequenceDescription of
Artificial Sequence Synthetic hybrid containing Phl p 5 MHC Class II
overlapping epitope 241Leu Arg Met Lys Xaa Tyr Glu Ser Tyr Lys Phe
Ile Pro Ala1 5 1024217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic hybrid
containing Phl p 5 MHC Class II overlapping epitope 242Leu Arg Met
Lys Xaa Tyr Glu Ser Tyr Lys Phe Ile Pro Ala Leu Glu1 5
10 15Ala24317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic hybrid
containing Phl p 5 MHC Class II overlapping epitope 243Leu Arg Met
Lys Xaa Leu Gln Val Ile Glu Lys Val Asp Ala Ala Phe1 5
10 15Lys24414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic hybrid
containing Phl p 5 MHC Class II overlapping epitope 244Leu Arg Met
Lys Xaa Phe Lys Val Ala Ala Thr Ala Ala Asn1 5
1024524PRTArtificial SequenceDescription of Artificial Sequence
Synthetic hybrid containing Phl p 5 MHC Class II overlapping
epitope 245Leu Arg Met Lys Xaa Leu Gln Val Ile Glu Lys Val Asp Ala Ala
Phe1 5 10 15Lys Val Ala
Ala Thr Ala Ala Asn 20246162PRTApis mellifera 246Gly Ser Leu
Phe Leu Leu Leu Leu Ser Thr Ser His Gly Trp Gln Ile1 5
10 15Arg Asp Arg Ile Gly Asp Asn Glu Leu
Glu Glu Arg Ile Ile Tyr Pro 20 25
30Gly Thr Leu Trp Cys Gly His Gly Asn Lys Ser Ser Gly Pro Asn Glu
35 40 45Leu Gly Arg Phe Lys His Thr
Asp Ala Cys Cys Arg Thr His Asp Met 50 55
60Cys Pro Asp Val Met Ser Ala Gly Glu Ser Lys His Gly Leu Thr Asn65
70 75 80Thr Ala Ser His
Thr Arg Leu Ser Cys Asp Cys Asp Asp Lys Phe Tyr 85
90 95Asp Cys Leu Lys Asn Ser Ala Asp Thr Ile
Ser Ser Tyr Phe Val Gly 100 105
110Lys Met Tyr Phe Asn Leu Ile Asp Thr Lys Cys Tyr Lys Leu Glu His
115 120 125Pro Val Thr Gly Cys Gly Glu
Arg Thr Glu Gly Arg Cys Leu His Tyr 130 135
140Thr Val Asp Lys Ser Lys Pro Lys Val Tyr Gln Trp Phe Asp Leu
Arg145 150 155 160Lys
Tyr2479PRTApis mellifera 247Trp Gln Ile Arg Asp Arg Ile Gly Asp1
52489PRTApis mellifera 248Phe Leu Leu Leu Leu Ser Thr Ser His1
52499PRTApis mellifera 249Phe Val Gly Lys Met Tyr Phe Asn Leu1
52509PRTApis mellifera 250Leu Ile Asp Thr Lys Cys Tyr Lys Leu1
52519PRTApis mellifera 251Leu Leu Leu Ser Thr Ser His Gly
Trp1 52529PRTApis mellifera 252Phe Asn Leu Ile Asp Thr Lys
Cys Tyr1 52539PRTApis mellifera 253Leu Leu Leu Leu Ser Thr
Ser His Gly1 52549PRTApis mellifera 254Phe Lys His Thr Asp
Ala Cys Cys Arg1 52559PRTApis mellifera 255Tyr Lys Leu Glu
His Pro Val Thr Gly1 525613PRTApis mellifera 256Lys Met Tyr
Phe Asn Leu Ile Asp Thr Lys Cys Tyr Lys1 5
1025713PRTApis mellifera 257Lys Cys Tyr Lys Leu Glu His Pro Val Thr Gly
Cys Gly1 5 1025813PRTApis mellifera
258Tyr Phe Val Gly Lys Met Tyr Phe Asn Leu Ile Asp Thr1 5
1025912PRTApis mellifera 259Cys Leu His Tyr Thr Val Asp
Lys Ser Lys Pro Lys1 5 1026018PRTApis
mellifera 260Glu Ser Lys His Gly Leu Thr Asn Thr Ala Ser His Thr Arg Leu
Ser1 5 10 15Cys
Asp26114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PHL A2 hybrid non-overlapping epitope 261Leu Arg
Met Lys Xaa Trp Gln Ile Arg Asp Arg Ile Gly Asp1 5
1026214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PHL A2 hybrid non-overlapping epitope 262Leu Arg
Met Lys Xaa Phe Lys His Thr Asp Ala Cys Cys Arg1 5
1026314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PHL A2 hybrid overlapping epitope 263Leu Arg Met
Lys Xaa Phe Leu Leu Leu Leu Ser Thr Ser His1 5
1026414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PHL A2 hybrid overlapping epitope 264Leu Arg Met
Lys Xaa Leu Leu Leu Leu Ser Thr Ser His Gly1 5
1026514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PHL A2 hybrid overlapping epitope 265Leu Arg Met
Lys Xaa Leu Leu Leu Ser Thr Ser His Gly Trp1 5
1026616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PHL A2 hybrid overlapping epitope 266Leu Arg Met
Lys Xaa Phe Leu Leu Leu Leu Ser Thr Ser His Gly Trp1 5
10 1526714PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/PHL A2 hybrid overlapping
epitope 267Leu Arg Met Lys Xaa Phe Val Gly Lys Met Tyr Phe Asn Leu1
5 1026814PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/PHL A2 hybrid overlapping
epitope 268Leu Arg Met Lys Xaa Phe Asn Leu Ile Asp Thr Lys Cys Tyr1
5 1026914PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/PHL A2 hybrid overlapping
epitope 269Leu Arg Met Lys Xaa Leu Ile Asp Thr Lys Cys Tyr Lys Leu1
5 1027022PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/PHL A2 hybrid overlapping
epitope 270Leu Arg Met Lys Xaa Phe Val Gly Lys Met Tyr Phe Asn Leu Ile
Asp1 5 10 15Thr Lys Cys
Tyr Lys Leu 20271204PRTBlattella germanica 271Met Ala Pro Ser
Tyr Lys Leu Thr Tyr Cys Pro Val Lys Ala Leu Gly1 5
10 15Glu Pro Ile Arg Phe Leu Leu Ser Tyr Gly
Glu Lys Asp Phe Glu Asp 20 25
30Tyr Arg Phe Gln Glu Gly Asp Trp Pro Asn Leu Lys Pro Ser Met Pro
35 40 45Phe Gly Lys Thr Pro Val Leu Glu
Ile Asp Gly Lys Gln Thr His Gln 50 55
60Ser Val Ala Ile Ser Arg Tyr Leu Gly Lys Gln Phe Gly Leu Ser Gly65
70 75 80Lys Asp Asp Trp Glu
Asn Leu Glu Ile Asp Met Ile Val Asp Thr Ile 85
90 95Ser Asp Phe Arg Ala Ala Ile Ala Asn Tyr His
Tyr Asp Ala Asp Glu 100 105
110Asn Ser Lys Gln Lys Lys Trp Asp Pro Leu Lys Lys Glu Thr Ile Pro
115 120 125Tyr Tyr Thr Lys Lys Phe Asp
Glu Val Val Lys Ala Asn Gly Gly Tyr 130 135
140Leu Ala Ala Gly Lys Leu Thr Trp Ala Asp Phe Tyr Phe Val Ala
Ile145 150 155 160Leu Asp
Tyr Leu Asn His Met Ala Lys Glu Asp Leu Val Ala Asn Gln
165 170 175Pro Asn Leu Lys Ala Leu Arg
Glu Lys Val Leu Gly Leu Pro Ala Ile 180 185
190Lys Ala Trp Val Ala Lys Arg Pro Pro Thr Asp Leu
195 2002729PRTBlattella germanica 272Phe Gly Lys Thr Pro
Val Leu Glu Ile1 52739PRTBlattella germanica 273Trp Val Ala
Lys Arg Pro Pro Thr Asp1 52749PRTBlattella germanica 274Met
Ile Val Asp Thr Ile Ser Asp Phe1 52759PRTBlattella
germanica 275Phe Tyr Phe Val Ala Ile Leu Asp Tyr1
52769PRTBlattella germanica 276Ile Arg Phe Leu Leu Ser Tyr Gly Glu1
52779PRTBlattella germanica 277Leu Glu Ile Asp Gly Lys Gln Thr
His1 52789PRTBlattella germanica 278Phe Arg Ala Ala Ile Ala
Asn Tyr His1 527920PRTBlattella germanica 279Ile Val Asp
Thr Ile Ser Asp Phe Arg Ala Ala Ile Ala Asn Tyr His1 5
10 15Tyr Asp Ala Asp
2028020PRTBlattella germanica 280Asp Tyr Leu Asn His Met Ala Lys Glu Asp
Leu Val Ala Asn Gln Pro1 5 10
15Asn Leu Lys Ala 202819PRTHomo sapiens 281Ile Pro Gln
Gln His Thr Gln Val Leu1 5282702PRTHomo sapiens 282Met Glu
Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln1 5
10 15Arg Leu Leu Leu Thr Ala Ser Leu
Leu Thr Phe Trp Asn Pro Pro Thr 20 25
30Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu
Gly 35 40 45Lys Glu Val Leu Leu
Leu Val His Asn Leu Pro Gln His Leu Phe Gly 50 55
60Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln
Ile Ile65 70 75 80Gly
Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser
85 90 95Gly Arg Glu Ile Ile Tyr Pro
Asn Ala Ser Leu Leu Ile Gln Asn Ile 100 105
110Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys
Ser Asp 115 120 125Leu Val Asn Glu
Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu 130
135 140Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro
Val Glu Asp Lys145 150 155
160Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr
165 170 175Leu Trp Trp Val Asn
Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln 180
185 190Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn
Val Thr Arg Asn 195 200 205Asp Thr
Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg 210
215 220Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr
Gly Pro Asp Ala Pro225 230 235
240Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn
245 250 255Leu Ser Cys His
Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe 260
265 270Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu
Leu Phe Ile Pro Asn 275 280 285Ile
Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser 290
295 300Asp Thr Gly Leu Asn Arg Thr Thr Val Thr
Thr Ile Thr Val Tyr Ala305 310 315
320Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val
Glu 325 330 335Asp Glu Asp
Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr 340
345 350Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser
Leu Pro Val Ser Pro Arg 355 360
365Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr 370
375 380Arg Asn Asp Val Gly Pro Tyr Glu
Cys Gly Ile Gln Asn Glu Leu Ser385 390
395 400Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu
Tyr Gly Pro Asp 405 410
415Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn
420 425 430Leu Ser Leu Ser Cys His
Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser 435 440
445Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu
Phe Ile 450 455 460Ser Asn Ile Thr Glu
Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn465 470
475 480Asn Ser Ala Ser Gly His Ser Arg Thr Thr
Val Lys Thr Ile Thr Val 485 490
495Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro
500 505 510Val Glu Asp Lys Asp
Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln 515
520 525Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser
Leu Pro Val Ser 530 535 540Pro Arg Leu
Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn545
550 555 560Val Thr Arg Asn Asp Ala Arg
Ala Tyr Val Cys Gly Ile Gln Asn Ser 565
570 575Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp
Val Leu Tyr Gly 580 585 590Pro
Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly 595
600 605Ala Asn Leu Asn Leu Ser Cys His Ser
Ala Ser Asn Pro Ser Pro Gln 610 615
620Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu625
630 635 640Phe Ile Ala Lys
Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe 645
650 655Val Ser Asn Leu Ala Thr Gly Arg Asn Asn
Ser Ile Val Lys Ser Ile 660 665
670Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr
675 680 685Val Gly Ile Met Ile Gly Val
Leu Val Gly Val Ala Leu Ile 690 695
7002839PRTHomo sapiens 283Tyr Arg Pro Gly Val Asn Leu Ser Leu1
52849PRTHomo sapiens 284Trp Val Asn Gly Gln Ser Leu Pro Val1
52859PRTHomo sapiens 285Trp Val Asn Asn Gln Ser Leu Pro Val1
52869PRTHomo sapiens 286Trp Arg Ile Asn Gly Ile Pro Gln Gln1
52879PRTHomo sapiens 287Tyr Arg Ser Gly Glu Asn Leu Asn Leu1
52889PRTHomo sapiens 288Leu Leu Leu Val His Asn Leu Pro Gln1
52899PRTHomo sapiens 289Trp Leu Ile Asp Gly Asn Ile Gln Gln1
52909PRTHomo sapiens 290Tyr Gly Pro Asp Thr Pro Ile Ile Ser1
52919PRTHomo sapiens 291Phe Tyr Thr Leu His Val Ile Lys Ser1
52929PRTHomo sapiens 292Ile Ile Gly Tyr Val Ile Gly Thr Gln1
529314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/CEA non-overlapping hybrid peptide 293Leu
Arg Met Lys Xaa Trp Val Asn Asn Gln Ser Leu Pro Val1 5
1029414PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/CEA non-overlapping hybrid peptide 294Leu
Arg Met Lys Xaa Tyr Arg Pro Gly Val Asn Leu Ser Leu1 5
1029514PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/CEA non-overlapping hybrid peptide 295Leu
Arg Met Lys Xaa Trp Arg Ile Asn Gly Ile Pro Gln Gln1 5
1029614PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/CEA non-overlapping hybrid peptide 296Leu
Arg Met Lys Xaa Tyr Arg Ser Gly Glu Asn Leu Asn Leu1 5
102979PRTHomo sapiens 297His Leu Phe Gly Tyr Ser Trp Tyr
Lys1 52989PRTHomo sapiens 298Thr Tyr Tyr Arg Pro Gly Val
Asn Leu1 52999PRTHomo sapiens 299Thr Tyr Ala Cys Phe Val
Ser Asn Leu1 53009PRTHomo sapiens 300Ile Met Ile Gly Val
Leu Val Gly Val1 53019PRTHomo sapiens 301Tyr Leu Ser Gly
Ala Asn Leu Asn Leu1 53029PRTHomo sapiens 302Ile Met Ile
Gly Val Leu Val Gly Val1 53039PRTHomo sapiens 303Leu Met
Thr Phe Trp Asn Pro Pro Val1 53049PRTHomo sapiens 304Tyr
Leu Ser Gly Ala Asn Leu Asn Leu1 530510PRTHomo sapiens
305Gln Tyr Ser Trp Phe Val Asn Gly Thr Phe1 5
103069PRTHomo sapiens 306Thr Tyr Ala Cys Phe Val Ser Asn Leu1
53078PRTHomo sapiens 307His Leu Phe Tyr Ser Trp Tyr Lys1
53089PRTHomo sapiens 308Leu Leu Thr Phe Trp Asn Pro Pro Val1
53099PRTHomo sapiens 309Leu Leu Thr Phe Trp Asn Pro Pro Thr1
531023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MHC Class II/MHC Class I CEA hybrid peptide
310Leu Arg Met Lys Xaa Trp Val Asn Asn Gln Ser Leu Pro Val Ile Met1
5 10 15Ile Gly Val Leu Val Gly
Val 2031116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/MHC Class II/MHC Class I CEA hybrid
peptide 311Leu Arg Met Lys Xaa Thr Tyr Tyr Arg Pro Gly Val Asn Leu Ser
Leu1 5 10
1531224PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/MHC Class II/MHC Class I CEA hybrid peptide 312Leu Arg
Met Lys Xaa Tyr Arg Ser Gly Glu Asn Leu Asn Leu Gln Tyr1 5
10 15Ser Trp Phe Val Asn Gly Thr Phe
2031322PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MHC Class II/MHC Class I CEA hybrid peptide
313Leu Arg Met Lys Xaa Leu Leu Leu Val His Asn Leu Pro Gln His Leu1
5 10 15Phe Tyr Ser Trp Tyr Lys
203141890PRTHomo sapiens 314Arg Val Asp Pro Ile Gly Pro Gly
Leu Asp Arg Glu Arg Leu Tyr Trp1 5 10
15Glu Leu Ser Gln Leu Thr Asn Ser Ile Thr Glu Leu Gly Pro
Tyr Thr 20 25 30Leu Asp Arg
Asp Ser Leu Tyr Val Asn Gly Phe Asn Pro Trp Ser Ser 35
40 45Val Pro Thr Thr Ser Thr Pro Gly Thr Ser Thr
Val His Leu Ala Thr 50 55 60Ser Gly
Thr Pro Ser Ser Leu Pro Gly His Thr Ala Pro Val Pro Leu65
70 75 80Leu Ile Pro Phe Thr Leu Asn
Phe Thr Ile Thr Asn Leu His Tyr Glu 85 90
95Glu Asn Met Gln His Pro Gly Ser Arg Lys Phe Asn Thr
Thr Glu Arg 100 105 110Val Leu
Gln Gly Leu Leu Lys Pro Leu Phe Lys Ser Thr Ser Val Gly 115
120 125Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu
Leu Arg Pro Glu Lys His 130 135 140Gly
Ala Ala Thr Gly Val Asp Ala Ile Cys Thr Leu Arg Leu Asp Pro145
150 155 160Thr Gly Pro Gly Leu Asp
Arg Glu Arg Leu Tyr Trp Glu Leu Ser Gln 165
170 175Leu Thr Asn Ser Val Thr Glu Leu Gly Pro Tyr Thr
Leu Asp Arg Asp 180 185 190Ser
Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser Val Pro Thr Thr 195
200 205Ser Ile Pro Gly Thr Ser Ala Val His
Leu Glu Thr Ser Gly Thr Pro 210 215
220Ala Ser Leu Pro Gly His Thr Ala Pro Gly Pro Leu Leu Val Pro Phe225
230 235 240Thr Leu Asn Phe
Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met Arg 245
250 255His Pro Gly Ser Arg Lys Phe Asn Thr Thr
Glu Arg Val Leu Gln Gly 260 265
270Leu Leu Lys Pro Leu Phe Lys Ser Thr Ser Val Gly Pro Leu Tyr Ser
275 280 285Gly Cys Arg Leu Thr Leu Leu
Arg Pro Glu Lys Arg Gly Ala Ala Thr 290 295
300Gly Val Asp Thr Ile Cys Thr His Arg Leu Asp Pro Leu Asn Pro
Gly305 310 315 320Leu Asp
Arg Glu Gln Leu Tyr Trp Glu Leu Ser Lys Leu Thr Arg Gly
325 330 335Ile Ile Glu Leu Gly Pro Tyr
Leu Leu Asp Arg Gly Ser Leu Tyr Val 340 345
350Asn Gly Phe Thr His Arg Asn Phe Val Pro Ile Thr Ser Thr
Pro Gly 355 360 365Thr Ser Thr Val
His Leu Gly Thr Ser Glu Thr Pro Ser Ser Leu Pro 370
375 380Arg Pro Ile Val Pro Gly Pro Leu Leu Val Pro Phe
Thr Leu Asn Phe385 390 395
400Thr Ile Thr Asn Leu Gln Tyr Glu Glu Ala Met Arg His Pro Gly Ser
405 410 415Arg Lys Phe Asn Thr
Thr Glu Arg Val Leu Gln Gly Leu Leu Arg Pro 420
425 430Leu Phe Lys Asn Thr Ser Ile Gly Pro Leu Tyr Ser
Ser Cys Arg Leu 435 440 445Thr Leu
Leu Arg Pro Glu Lys Asp Lys Ala Ala Thr Arg Val Asp Ala 450
455 460Ile Cys Thr His His Pro Asp Pro Gln Ser Pro
Gly Leu Asn Arg Glu465 470 475
480Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Gly Ile Thr Glu Leu
485 490 495Gly Pro Tyr Thr
Leu Asp Arg Asp Ser Leu Tyr Val Asp Gly Phe Thr 500
505 510His Trp Ser Pro Ile Pro Thr Thr Ser Thr Pro
Gly Thr Ser Ile Val 515 520 525Asn
Leu Gly Thr Ser Gly Ile Pro Pro Ser Leu Pro Glu Thr Thr Ala 530
535 540Thr Gly Pro Leu Leu Val Pro Phe Thr Leu
Asn Phe Thr Ile Thr Asn545 550 555
560Leu Gln Tyr Glu Glu Asn Met Gly His Pro Gly Ser Arg Lys Phe
Asn 565 570 575Ile Thr Glu
Ser Val Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys Ser 580
585 590Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys
Arg Leu Thr Leu Leu Arg 595 600
605Pro Glu Lys Asp Gly Val Ala Thr Arg Val Asp Ala Ile Cys Thr His 610
615 620Arg Pro Asp Pro Lys Ile Pro Gly
Leu Asp Arg Gln Gln Leu Tyr Trp625 630
635 640Glu Leu Ser Gln Leu Thr His Ser Ile Thr Glu Leu
Gly Pro Tyr Thr 645 650
655Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr Gln Arg Ser Ser
660 665 670Val Pro Thr Thr Ser Thr
Pro Gly Thr Phe Thr Val Gln Pro Glu Thr 675 680
685Ser Glu Thr Pro Ser Ser Leu Pro Gly Pro Thr Ala Thr Gly
Pro Val 690 695 700Leu Leu Pro Phe Thr
Leu Asn Phe Thr Ile Ile Asn Leu Gln Tyr Glu705 710
715 720Glu Asp Met His Arg Pro Gly Ser Arg Lys
Phe Asn Thr Thr Glu Arg 725 730
735Val Leu Gln Gly Leu Leu Met Pro Leu Phe Lys Asn Thr Ser Val Ser
740 745 750Ser Leu Tyr Ser Gly
Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp 755
760 765Gly Ala Ala Thr Arg Val Asp Ala Val Cys Thr His
Arg Pro Asp Pro 770 775 780Lys Ser Pro
Gly Leu Asp Arg Glu Arg Leu Tyr Trp Lys Leu Ser Gln785
790 795 800Leu Thr His Gly Ile Thr Glu
Leu Gly Pro Tyr Thr Leu Asp Arg His 805
810 815Ser Leu Tyr Val Asn Gly Phe Thr His Gln Ser Ser
Met Thr Thr Thr 820 825 830Arg
Thr Pro Asp Thr Ser Thr Met His Leu Ala Thr Ser Arg Thr Pro 835
840 845Ala Ser Leu Ser Gly Pro Thr Thr Ala
Ser Pro Leu Leu Val Leu Phe 850 855
860Thr Ile Asn Phe Thr Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met His865
870 875 880His Pro Gly Ser
Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly 885
890 895Leu Leu Arg Pro Val Phe Lys Asn Thr Ser
Val Gly Pro Leu Tyr Ser 900 905
910Gly Cys Arg Leu Thr Leu Leu Arg Pro Lys Lys Asp Gly Ala Ala Thr
915 920 925Lys Val Asp Ala Ile Cys Thr
Tyr Arg Pro Asp Pro Lys Ser Pro Gly 930 935
940Leu Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His
Ser945 950 955 960Ile Thr
Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val
965 970 975Asn Gly Phe Thr Gln Arg Ser
Ser Val Pro Thr Thr Ser Ile Pro Gly 980 985
990Thr Pro Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Val Ser
Lys Pro 995 1000 1005Gly Pro Ser Ala
Ala Ser Pro Leu Leu Val Leu Phe Thr Leu Asn Phe 1010
1015 1020Thr Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met Gln
His Pro Gly Ser1025 1030 1035
1040Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Arg Ser
1045 1050 1055Leu Phe Lys Ser Thr
Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu 1060
1065 1070Thr Leu Leu Arg Pro Glu Lys Asp Gly Thr Ala Thr
Gly Val Asp Ala 1075 1080 1085Ile
Cys Thr His His Pro Asp Pro Lys Ser Pro Arg Leu Asp Arg Glu 1090
1095 1100Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr
His Asn Ile Thr Glu Leu1105 1110 1115
1120Gly Pro Tyr Ala Leu Asp Asn Asp Ser Leu Phe Val Asn Gly Phe
Thr 1125 1130 1135His Arg
Ser Ser Val Ser Thr Thr Ser Thr Pro Gly Thr Pro Thr Val 1140
1145 1150Tyr Leu Gly Ala Ser Lys Thr Pro Ala
Ser Ile Phe Gly Pro Ser Ala 1155 1160
1165Ala Ser His Leu Leu Ile Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn
1170 1175 1180Leu Arg Tyr Glu Glu Asn Met
Trp Pro Gly Ser Arg Lys Phe Asn Thr1185 1190
1195 1200Thr Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Leu
Phe Lys Asn Thr 1205 1210
1215Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro
1220 1225 1230Glu Lys Asp Gly Glu Ala
Thr Gly Val Asp Ala Ile Cys Thr His Arg 1235 1240
1245Pro Asp Pro Thr Gly Pro Gly Leu Asp Arg Glu Gln Leu Tyr
Leu Glu 1250 1255 1260Leu Ser Gln Leu
Thr His Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu1265 1270
1275 1280Asp Arg Asp Ser Leu Tyr Val Asn Gly
Phe Thr His Arg Ser Ser Val 1285 1290
1295Pro Thr Thr Ser Thr Gly Val Val Ser Glu Glu Pro Phe Thr Leu
Asn 1300 1305 1310Phe Thr Ile
Asn Asn Leu Arg Tyr Met Ala Asp Met Gly Gln Pro Gly 1315
1320 1325Ser Leu Lys Phe Asn Ile Thr Asp Asn Val Met
Gln His Leu Leu Ser 1330 1335 1340Pro
Leu Phe Gln Arg Ser Ser Leu Gly Ala Arg Tyr Thr Gly Cys Arg1345
1350 1355 1360Val Ile Ala Leu Arg Ser
Val Lys Asn Gly Ala Glu Thr Arg Val Asp 1365
1370 1375Leu Leu Cys Thr Tyr Leu Gln Pro Leu Ser Gly Pro
Gly Leu Pro Ile 1380 1385
1390Lys Gln Val Phe His Glu Leu Ser Gln Gln Thr His Gly Ile Thr Arg
1395 1400 1405Leu Gly Pro Tyr Ser Leu Asp
Lys Asp Ser Leu Tyr Leu Asn Gly Tyr 1410 1415
1420Asn Glu Pro Gly Pro Asp Glu Pro Pro Thr Thr Pro Lys Pro Ala
Thr1425 1430 1435 1440Thr Phe
Leu Pro Pro Leu Ser Glu Ala Thr Thr Ala Met Gly Tyr His
1445 1450 1455Leu Lys Thr Leu Thr Leu Asn
Phe Thr Ile Ser Asn Leu Gln Tyr Ser 1460 1465
1470Pro Asp Met Gly Lys Gly Ser Ala Thr Phe Asn Ser Thr Glu
Gly Val 1475 1480 1485Leu Gln His
Leu Leu Arg Pro Leu Phe Gln Lys Ser Ser Met Gly Pro 1490
1495 1500Phe Tyr Leu Gly Cys Gln Leu Ile Ser Leu Arg Pro
Glu Lys Asp Gly1505 1510 1515
1520Ala Ala Thr Gly Val Asp Thr Thr Cys Thr Tyr His Pro Asp Pro Val
1525 1530 1535Gly Pro Gly Leu Asp
Ile Gln Gln Leu Tyr Trp Glu Leu Ser Gln Leu 1540
1545 1550Thr His Gly Val Thr Gln Leu Gly Phe Tyr Val Leu
Asp Arg Asp Ser 1555 1560 1565Leu
Phe Ile Asn Gly Tyr Ala Pro Gln Asn Leu Ser Ile Arg Gly Glu 1570
1575 1580Tyr Gln Ile Asn Phe His Ile Val Asn Trp
Asn Leu Ser Asn Pro Asp1585 1590 1595
1600Pro Thr Ser Ser Glu Tyr Ile Thr Leu Leu Arg Asp Ile Gln Asp
Lys 1605 1610 1615Val Thr
Thr Leu Tyr Lys Gly Ser Gln Leu His Asp Thr Phe Arg Phe 1620
1625 1630Cys Leu Val Thr Asn Leu Thr Met Asp
Ser Val Leu Val Thr Val Lys 1635 1640
1645Ala Leu Phe Ser Ser Asn Leu Asp Pro Ser Leu Val Glu Gln Val Phe
1650 1655 1660Leu Asp Lys Thr Leu Asn Ala
Ser Phe His Trp Leu Gly Ser Thr Tyr1665 1670
1675 1680Gln Leu Val Asp Ile His Val Thr Glu Met Glu Ser
Ser Val Tyr Gln 1685 1690
1695Pro Thr Ser Ser Ser Ser Thr Gln His Phe Tyr Pro Asn Phe Thr Ile
1700 1705 1710Thr Asn Leu Pro Tyr Ser
Gln Asp Lys Ala Gln Pro Gly Thr Thr Asn 1715 1720
1725Tyr Gln Arg Asn Lys Arg Asn Ile Glu Asp Ala Leu Asn Gln
Leu Phe 1730 1735 1740Arg Asn Ser Ser
Ile Lys Ser Tyr Phe Ser Asp Cys Gln Val Ser Thr1745 1750
1755 1760Phe Arg Ser Val Pro Asn Arg His His
Thr Gly Val Asp Ser Leu Cys 1765 1770
1775Asn Phe Ser Pro Leu Ala Arg Arg Val Asp Arg Val Ala Ile Tyr
Glu 1780 1785 1790Glu Phe Leu
Arg Met Thr Arg Asn Gly Thr Gln Leu Gln Asn Phe Thr 1795
1800 1805Leu Asp Arg Ser Ser Val Leu Val Asp Gly Tyr
Ser Pro Asn Arg Asn 1810 1815 1820Glu
Pro Leu Thr Gly Asn Ser Asp Leu Pro Phe Trp Ala Val Ile Leu1825
1830 1835 1840Ile Gly Leu Ala Gly Leu
Leu Gly Leu Ile Thr Cys Leu Ile Cys Gly 1845
1850 1855Val Leu Val Thr Thr Arg Arg Arg Lys Lys Glu Gly
Glu Tyr Asn Val 1860 1865
1870Gln Gln Gln Cys Pro Gly Tyr Tyr Gln Ser His Leu Asp Leu Glu Asp
1875 1880 1885Leu Gln18903159PRTHomo
sapiens 315Phe Arg Phe Cys Leu Val Thr Asn Leu1
53169PRTHomo sapiens 316Val Leu Phe Thr Leu Asn Phe Thr Ile1
53179PRTHomo sapiens 317Ile Leu Phe Thr Leu Asn Phe Thr Ile1
53189PRTHomo sapiens 318Leu Arg Leu Asp Pro Thr Gly Pro Gly1
53199PRTHomo sapiens 319Leu Val Leu Phe Thr Leu Asn Phe Thr1
53209PRTHomo sapiens 320Leu Val Leu Phe Thr Ile Asn Phe Thr1
53219PRTHomo sapiens 321Ile Val Asn Leu Gly Thr Ser Gly Ile1
53229PRTHomo sapiens 322Leu Arg Tyr Met Ala Asp Met Gly Gln1
53239PRTHomo sapiens 323Leu Arg Tyr Glu Glu Asn Met Gln His1
53249PRTHomo sapiens 324Leu Arg Tyr Glu Glu Asn Met His His1
53259PRTHomo sapiens 325Val Phe Leu Asp Lys Thr Leu Asn Ala1
53269PRTHomo sapiens 326Leu Leu Ile Leu Phe Thr Leu Asn Phe1
53279PRTHomo sapiens 327Tyr Arg Pro Asp Pro Lys Ser Pro Gly1
53289PRTHomo sapiens 328Leu Val Pro Phe Thr Leu Asn Phe Thr1
53299PRTHomo sapiens 329Leu Arg Pro Leu Phe Lys Asn Thr Ser1
53309PRTHomo sapiens 330Leu Arg Tyr Glu Glu Asn Met Trp
Pro1 53319PRTHomo sapiens 331Leu Val Thr Asn Leu Thr Met
Asp Ser1 53329PRTHomo sapiens 332Phe Val Pro Ile Thr Ser
Thr Pro Gly1 53339PRTHomo sapiens 333Leu Arg Pro Leu Phe
Lys Asn Thr Ser1 53349PRTHomo sapiens 334Leu Arg Pro Val
Phe Lys Asn Thr Ser1 53359PRTHomo sapiens 335Tyr His Pro
Asp Pro Val Gly Pro Gly1 533614PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/CA125
conserved tandem-repeat hybrid peptide 336Leu Arg Met Lys Xaa Val Leu Phe
Thr Leu Asn Phe Thr Ile1 5
1033714PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/CA125 conserved tandem-repeat hybrid peptide 337Leu Arg Met
Lys Xaa Ile Leu Phe Thr Leu Asn Phe Thr Ile1 5
1033814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/CA125 conserved tandem-repeat hybrid peptide 338Leu
Arg Met Lys Xaa Leu Val Leu Phe Thr Ile Asn Phe Thr1 5
1033914PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/CA125 conserved tandem-repeat hybrid
peptide 339Leu Arg Met Lys Xaa Leu Arg Tyr Glu Glu Asn Met Gln His1
5 1034014PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/CA125 conserved tandem-repeat
hybrid peptide 340Leu Arg Met Lys Xaa Leu Arg Tyr Glu Glu Asn Met His
His1 5 1034114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/CA125
conserved tandem-repeat hybrid peptide 341Leu Arg Met Lys Xaa Leu Arg Tyr
Glu Glu Asn Met Trp Pro1 5
1034214PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/CA125 overlapping MHC II hybrid peptide 342Leu Arg Met Lys
Xaa Phe Arg Phe Cys Leu Val Thr Asn Leu1 5
1034314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/CA125 overlapping MHC II hybrid peptide 343Leu Arg
Met Lys Xaa Leu Val Thr Asn Leu Thr Met Asp Ser1 5
1034418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/CA125 overlapping MHC II hybrid peptide 344Leu Arg
Met Lys Xaa Phe Arg Phe Cys Leu Val Thr Asn Leu Thr Met1 5
10 15Asp Ser3459PRTHomo sapiens 345Trp
Leu Gly Ser Thr Tyr Gln Leu Val1 53469PRTHomo sapiens
346Val Leu Phe Thr Leu Asn Phe Thr Ile1 53479PRTHomo
sapiens 347Ile Leu Phe Thr Leu Asn Phe Thr Ile1
53489PRTHomo sapiens 348Val Leu Phe Thr Ile Asn Phe Thr Ile1
53499PRTHomo sapiens 349Leu Leu Asp Arg Gly Ser Leu Tyr Val1
53509PRTHomo sapiens 350Tyr Leu Gly Cys Gln Leu Ile Ser Leu1
53519PRTHomo sapiens 351Thr Leu Asn Ala Ser Phe His Trp Leu1
53529PRTHomo sapiens 352Gly Val Thr Gln Leu Gly Phe Tyr Val1
53539PRTHomo sapiens 353Gly Leu Leu Gly Leu Ile Thr Cys Leu1
53549PRTHomo sapiens 354Lys Leu Thr Arg Gly Ile Ile Glu Leu1
535514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MHC II epitope/MHC I epitope hybrid peptide
355Leu Arg Met Lys Xaa Phe Arg Phe Cys Leu Val Thr Asn Leu1
5 1035615PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/MHC II epitope/MHC I epitope
hybrid peptide 356Leu Arg Met Lys Xaa Leu Val Pro Phe Thr Leu Asn
Phe Thr Ile1 5 10
153576PRTArtificial SequenceDescription of Artificial Sequence Synthetic
6X-His tag 357His His His His His His1 5358261PRTHomo
sapiens 358Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile
Gly1 5 10 15Ala Ala Pro
Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20
25 30Lys His Ser Gln Pro Trp Gln Val Leu Val
Ala Ser Arg Gly Arg Ala 35 40
45Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala 50
55 60His Cys Ile Arg Asn Lys Ser Val Ile
Leu Leu Gly Arg His Ser Leu65 70 75
80Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His
Ser Phe 85 90 95Pro His
Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg 100
105 110Pro Gly Asp Asp Ser Ser His Asp Leu
Met Leu Leu Arg Leu Ser Glu 115 120
125Pro Ala Glu Leu Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln
130 135 140Glu Pro Ala Leu Gly Thr Thr
Cys Tyr Ala Ser Gly Trp Gly Ser Ile145 150
155 160Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln
Cys Val Asp Leu 165 170
175His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val
180 185 190Thr Lys Phe Met Leu Cys
Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr 195 200
205Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val
Leu Gln 210 215 220Gly Ile Thr Ser Trp
Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro225 230
235 240Ser Leu Tyr Thr Lys Val Val His Tyr Arg
Lys Trp Ile Lys Asp Thr 245 250
255Ile Val Ala Asn Pro 2603599PRTHomo sapiens 359Trp Val
Leu Thr Ala Ala His Cys Ile1 53609PRTHomo sapiens 360Trp
Val Pro Val Val Phe Leu Thr Leu1 53619PRTHomo sapiens
361Leu Arg Leu Ser Glu Pro Ala Glu Leu1 53629PRTHomo
sapiens 362Ile Arg Asn Lys Ser Val Ile Leu Leu1
53639PRTHomo sapiens 363Leu Leu Gly Arg His Ser Leu Phe His1
53649PRTHomo sapiens 364Val Ile Leu Leu Gly Arg His Ser Leu1
53659PRTHomo sapiens 365Leu Gln Gly Ile Thr Ser Trp Gly Ser1
536616PRTHomo sapiens 366Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His
Tyr Arg Lys Trp Ile1 5 10
1536714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PSA non-overlapping hybrid peptide 367Leu Arg Met
Lys Xaa Trp Val Pro Val Val Phe Leu Thr Leu1 5
1036814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PSA non-overlapping hybrid peptide 368Leu Arg Met
Lys Xaa Leu Arg Leu Ser Glu Pro Ala Glu Leu1 5
1036914PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PSA non-overlapping hybrid peptide 369Leu Arg Met
Lys Xaa Leu Gln Gly Ile Thr Ser Trp Gly Ser1 5
1037014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PSA overlapping hybrid peptide 370Leu Arg Met Lys
Xaa Trp Val Leu Thr Ala Ala His Cys Ile1 5
1037114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PSA overlapping hybrid peptide 371Leu Arg Met Lys
Xaa Ile Arg Asn Lys Ser Val Ile Leu Leu1 5
1037214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PSA overlapping hybrid peptide 372Leu Arg Met Lys
Xaa Val Ile Leu Leu Gly Arg His Ser Leu1 5
1037314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PSA overlapping hybrid peptide 373Leu Arg Met Lys
Xaa Leu Leu Gly Arg His Ser Leu Phe His1 5
1037429PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/PSA overlapping hybrid peptide 374Leu Arg Met Lys
Xaa Trp Val Leu Thr Ala Ala His Cys Ile Arg Asn1 5
10 15Lys Ser Val Ile Leu Leu Gly Arg His Ser
Leu Phe His 20 253758PRTHomo sapiens 375Gly
Arg Ala Val Cys Gly Val Leu1 53769PRTHomo sapiens 376Ile
Arg Asn Lys Ser Val Ile Leu Leu1 53779PRTHomo sapiens
377Leu Arg Leu Ser Glu Pro Ala Glu Leu1 53789PRTHomo
sapiens 378Ala Pro Leu Ile Leu Ser Arg Ile Val1
53799PRTHomo sapiens 379Phe Leu Thr Leu Ser Val Thr Trp Ile1
53809PRTHomo sapiens 380Tyr Arg Lys Trp Ile Lys Asp Thr Ile1
538110PRTHomo sapiens 381His Pro Gln Lys Val Thr Lys Phe Met Leu1
5 1038219PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/PSA MHC II-presented
epitope/PSA MHC I- presented overlapping hybrid peptide 382Leu Arg
Met Lys Xaa Trp Val Pro Val Val Phe Leu Thr Leu Ser Val1 5
10 15Thr Trp Ile38314PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/PSA MHC
II-presented epitope/PSA MHC I- presented overlapping hybrid peptide
383Leu Arg Met Lys Xaa Ile Arg Asn Lys Ser Val Ile Leu Leu1
5 1038414PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/PSA MHC II-presented
epitope/PSA MHC I- presented overlapping hybrid peptide 384Leu Arg
Met Lys Xaa Leu Arg Leu Ser Glu Pro Ala Glu Leu1 5
10385660PRTHomo sapiens 385Met Asp Leu Val Leu Lys Arg Cys Leu
Leu His Leu Ala Val Ile Gly1 5 10
15Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pro Arg Asn Gln Asp
Trp 20 25 30Leu Gly Val Ser
Arg Gln Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu 35
40 45Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys
Trp Arg Gly Gly 50 55 60Gln Val Ser
Leu Lys Val Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala65 70
75 80Asn Ala Ser Phe Ser Ile Ala Leu
Asn Phe Pro Gly Ser Gln Lys Val 85 90
95Leu Pro Asp Gly Val Ile Trp Val Asn Asn Thr Ile Ile Asn
Gly Ser 100 105 110Gln Val Trp
Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp Ala 115
120 125Cys Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser
Gly Ser Trp Ser Gln 130 135 140Lys Arg
Ser Phe Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln145
150 155 160Val Leu Gly Gly Pro Val Ser
Gly Leu Ser Ile Gly Thr Gly Arg Ala 165
170 175Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr
His Arg Arg Gly 180 185 190Ser
Arg Ser Tyr Val Pro Leu Ala His Ser Ser Ser Ala Phe Thr Ile 195
200 205Thr Asp Gln Val Pro Phe Ser Val Ser
Val Ser Gln Leu Arg Ala Leu 210 215
220Asp Gly Gly Asn Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe Ala225
230 235 240Leu Gln Leu His
Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu Ser 245
250 255Tyr Thr Trp Asp Phe Gly Asp Ser Ser Gly
Thr Leu Ile Ser Arg Ala 260 265
270Leu Val Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala Gln
275 280 285Val Val Leu Gln Ala Ala Ile
Pro Leu Thr Ser Cys Gly Ser Ser Pro 290 295
300Val Pro Gly Thr Thr Asp Gly His Arg Pro Thr Ala Glu Ala Pro
Asn305 310 315 320Thr Thr
Ala Gly Gln Val Pro Thr Thr Glu Val Val Gly Thr Thr Pro
325 330 335Gly Gln Ala Pro Thr Ala Glu
Pro Ser Gly Thr Thr Ser Val Gln Val 340 345
350Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro
Thr Ala 355 360 365Glu Ser Thr Gly
Met Thr Pro Glu Lys Val Pro Val Ser Glu Val Met 370
375 380Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala
Thr Gly Met Thr385 390 395
400Pro Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala Gln
405 410 415Val Thr Thr Thr Glu
Trp Val Glu Thr Thr Ala Arg Glu Leu Pro Ile 420
425 430Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met
Ser Thr Glu Ser 435 440 445Ile Thr
Gly Ser Leu Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu Arg 450
455 460Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val
Leu Tyr Arg Tyr Gly465 470 475
480Ser Phe Ser Val Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala Glu
485 490 495Ile Leu Gln Ala
Val Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu Thr 500
505 510Val Ser Cys Gln Gly Gly Leu Pro Lys Glu Ala
Cys Met Glu Ile Ser 515 520 525Ser
Pro Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val Leu 530
535 540Pro Ser Pro Ala Cys Gln Leu Val Leu His
Gln Ile Leu Lys Gly Gly545 550 555
560Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser
Leu 565 570 575Ala Val Val
Ser Thr Gln Leu Ile Met Pro Gly Gln Glu Ala Gly Leu 580
585 590Gly Gln Val Pro Leu Ile Val Gly Ile Leu
Leu Val Leu Met Ala Val 595 600
605Val Leu Ala Ser Leu Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp Phe 610
615 620Ser Val Pro Gln Leu Pro His Ser
Ser Ser His Trp Leu Arg Leu Pro625 630
635 640Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn Ser
Pro Leu Leu Ser 645 650
655Gly Gln Gln Val 6603869PRTHomo sapiens 386Val Tyr Val Trp
Lys Thr Trp Gly Gln1 53878PRTHomo sapiens 387Trp Val Glu
Thr Thr Arg Glu Leu1 53889PRTHomo sapiens 388Leu Tyr Arg
Tyr Gly Ser Phe Ser Val1 53899PRTHomo sapiens 389Val Val
Leu Gln Ala Ala Ile Pro Leu1 53909PRTHomo sapiens 390Leu
Val Leu His Gln Ile Leu Lys Gly1 53919PRTHomo sapiens
391Val Pro Leu Ile Val Gly Ile Leu Leu1 53929PRTHomo
sapiens 392Val Gly Ile Leu Leu Val Leu Met Ala1
53939PRTHomo sapiens 393Val Leu Met Ala Val Val Leu Ala Ser1
53949PRTHomo sapiens 394Leu Val Leu Met Ala Val Val Leu Ala1
53959PRTHomo sapiens 395Leu Val Leu Lys Arg Cys Leu Leu His1
53969PRTHomo sapiens 396Ile Tyr Arg Arg Arg Leu Met Lys Gln1
53979PRTHomo sapiens 397Tyr Arg Arg Arg Leu Met Lys Gln Asp1
53989PRTHomo sapiens 398Leu Tyr Pro Glu Trp Thr Glu Ala Gln1
539916PRTHomo sapiens 399Trp Asn Arg Gln Leu Tyr Pro Glu Trp Thr
Glu Ala Gln Arg Leu Asp1 5 10
1540019PRTHomo sapiens 400Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp
Phe Ser Val Pro Gln Leu1 5 10
15Pro His Ser40113PRTHomo sapiens 401Ser Leu Ala Val Val Ser Thr Gln
Leu Ile Met Pro Gly1 5 1040215PRTHomo
sapiens 402Gly Arg Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr1
5 10 1540316PRTHomo
sapiens 403Gly Pro Thr Leu Ile Gly Ala Asn Ala Ser Phe Ser Ile Ala Leu
Asn1 5 10
1540424PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 non-overlapping hybrid peptide 404Leu Arg Met Lys Xaa
Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp Phe1 5
10 15Ser Val Pro Gln Leu Pro His Ser
2040514PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 non-overlapping hybrid peptide 405Leu Arg Met Lys Xaa
Leu Val Leu Lys Arg Cys Leu Leu His1 5
1040614PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 non-overlapping hybrid peptide 406Leu Arg Met Lys Xaa
Val Tyr Val Trp Lys Thr Trp Gly Gln1 5
1040714PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 non-overlapping hybrid peptide 407Leu Arg Met Lys Xaa
Trp Val Glu Thr Thr Ala Arg Glu Leu1 5
1040814PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 non-overlapping hybrid peptide 408Leu Arg Met Lys Xaa
Leu Tyr Arg Tyr Gly Ser Phe Ser Val1 5
1040921PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 overlapping hybrid peptide 409Leu Arg Met Lys Xaa Trp
Asn Arg Gln Leu Tyr Pro Glu Trp Thr Glu1 5
10 15Ala Gln Arg Leu Asp
2041014PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 overlapping hybrid peptide 410Leu Arg Met Lys Xaa Leu
Tyr Pro Glu Trp Thr Glu Ala Gln1 5
1041121PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 overlapping hybrid peptide 411Leu Arg Met Lys Xaa Trp
Asn Arg Gln Leu Tyr Pro Glu Trp Thr Glu1 5
10 15Ala Gln Arg Leu Asp
2041214PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 overlapping hybrid peptide 412Leu Arg Met Lys Xaa Val
Pro Leu Ile Val Gly Ile Leu Leu1 5
1041314PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 overlapping hybrid peptide 413Leu Arg Met Lys Xaa Val
Gly Ile Leu Leu Val Leu Met Ala1 5
1041414PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 overlapping hybrid peptide 414Leu Arg Met Lys Xaa Val
Leu Met Ala Val Val Leu Ala Ser1 5
1041523PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 overlapping hybrid peptide 415Leu Arg Met Lys Xaa Val
Pro Leu Ile Val Gly Ile Leu Leu Val Leu1 5
10 15Met Ala Val Val Leu Ala Ser
204169PRTHomo sapiens 416Arg Leu Met Lys Gln Asp Phe Ser Val1
54179PRTHomo sapiens 417Leu Pro Lys Glu Ala Cys Met Glu Ile1
54189PRTHomo sapiens 418Ile Leu Leu Val Leu Met Ala Val Val1
54199PRTHomo sapiens 419Arg Tyr Gly Ser Phe Ser Val Thr Leu1
54209PRTHomo sapiens 420Lys Thr Trp Gly Gln Tyr Trp Gln Val1
54219PRTHomo sapiens 421Ala Leu Leu Ala Val Gly Ala Thr Lys1
54229PRTHomo sapiens 422Leu Ile Tyr Arg Arg Arg Leu Met Lys1
54239PRTHomo sapiens 423Tyr Leu Glu Pro Gly Pro Val Thr Ala1
54249PRTHomo sapiens 424Ala Leu Leu Ala Val Gly Ala Thr Lys1
54259PRTHomo sapiens 425Ile Thr Asp Gln Val Pro Phe Ser Val1
54269PRTHomo sapiens 426Leu Ile Tyr Arg Arg Arg Leu Met Lys1
54279PRTHomo sapiens 427Arg Leu Met Lys Gln Asp Phe Ser Val1
54289PRTHomo sapiens 428Arg Leu Pro Arg Ile Phe Cys Ser Cys1
54299PRTHomo sapiens 429Lys Thr Trp Gly Gln Tyr Trp Gln
Val1 543010PRTHomo sapiens 430Ala Met Leu Gly Thr His Thr
Met Glu Val1 5 1043110PRTHomo sapiens
431Ser Leu Ala Asp Thr Asn Ser Leu Ala Val1 5
104329PRTHomo sapiens 432Val Ser Asn Asp Gly Pro Thr Leu Ile1
54339PRTHomo sapiens 433Ala Leu Asn Phe Pro Gly Ser Gln Lys1
543423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/gp 100 non-overlapping hybrid peptide 434Leu Arg
Met Lys Xaa Leu Pro Lys Glu Ala Cys Met Glu Ile Leu Val1 5
10 15Leu His Gln Ile Leu Lys Gly
2043523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/gp 100 non-overlapping hybrid peptide 435Leu Arg
Met Lys Xaa Ala Leu Leu Ala Val Gly Ala Thr Lys Leu Val1 5
10 15Leu Lys Arg Cys Leu Leu His
2043624PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/gp 100 overlapping hybrid peptide 436Leu Arg Met
Lys Xaa Ser Leu Ala Asp Thr Asn Ser Leu Ala Val Val1 5
10 15Ser Thr Gln Leu Ile Met Pro Gly
2043720PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/gp 100 overlapping hybrid peptide 437Leu Arg Met
Lys Xaa Gly Arg Ala Met Leu Gly Thr His Thr Met Glu1 5
10 15Val Thr Val Tyr
2043831PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 overlapping hybrid peptide 438Leu Arg Met Lys Xaa Val
Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala1 5
10 15Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro Gly
Ser Gln Lys 20 25
3043925PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/gp 100 overlapping hybrid peptide 439Leu Arg Met Lys Xaa Leu
Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp1 5
10 15Phe Ser Val Pro Gln Leu Pro His Ser 20
2544017PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/gp 100 overlapping hybrid peptide 440Leu
Arg Met Lys Xaa Val Tyr Val Lys Thr Trp Gly Gln Tyr Trp Gln1
5 10 15Val44116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/gp 100
overlapping hybrid peptide 441Leu Arg Met Lys Xaa Leu Tyr Arg Tyr Gly Ser
Phe Ser Val Thr Leu1 5 10
15442519PRTHomo sapiens 442Met Ser Pro Leu Trp Trp Gly Phe Leu Leu Ser
Cys Leu Gly Cys Lys1 5 10
15Ile Leu Pro Gly Ala Gln Gly Gln Phe Pro Arg Val Cys Met Thr Val
20 25 30Asp Ser Leu Val Asn Lys Glu
Cys Cys Pro Arg Leu Gly Ala Glu Ser 35 40
45Ala Asn Val Cys Gly Ser Gln Gln Gly Arg Gly Gln Cys Thr Glu
Val 50 55 60Arg Ala Asp Thr Arg Pro
Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln65 70
75 80Asp Asp Arg Glu Leu Trp Pro Arg Lys Phe Phe
His Arg Thr Cys Lys 85 90
95Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Asp Cys Lys Phe Gly
100 105 110Trp Thr Gly Pro Asn Cys
Glu Arg Lys Lys Pro Pro Val Ile Arg Gln 115 120
125Asn Ile His Ser Leu Ser Pro Gln Glu Arg Glu Gln Phe Leu
Gly Ala 130 135 140Leu Asp Leu Ala Lys
Lys Arg Val His Pro Asp Tyr Val Ile Thr Thr145 150
155 160Gln His Trp Leu Gly Leu Leu Gly Pro Asn
Gly Thr Gln Pro Gln Phe 165 170
175Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser
180 185 190Val Arg Asp Thr Leu
Leu Gly Pro Gly Arg Pro Tyr Arg Ala Ile Asp 195
200 205Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His
Arg Tyr His Leu 210 215 220Leu Cys Leu
Glu Arg Asp Leu Gln Arg Leu Ile Gly Asn Glu Ser Phe225
230 235 240Ala Leu Pro Tyr Trp Asn Phe
Ala Thr Gly Arg Asn Glu Cys Asp Val 245
250 255Cys Thr Asp Gln Leu Phe Gly Ala Ala Arg Pro Asp
Asp Pro Thr Leu 260 265 270Ile
Ser Arg Asn Ser Arg Phe Ser Ser Trp Glu Thr Val Cys Asp Ser 275
280 285Leu Asp Asp Tyr Asn His Leu Val Thr
Leu Cys Asn Gly Thr Tyr Glu 290 295
300Gly Leu Leu Arg Arg Asn Gln Met Gly Arg Asn Ser Met Lys Leu Pro305
310 315 320Thr Leu Lys Asp
Ile Arg Asp Cys Leu Ser Leu Gln Lys Phe Asp Asn 325
330 335Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser
Phe Arg Asn Ala Leu Glu 340 345
350Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met Ser Leu
355 360 365His Asn Leu Val His Ser Phe
Leu Asn Gly Thr Asn Ala Leu Pro His 370 375
380Ser Ala Ala Asn Asp Pro Ile Phe Val Val Leu His Ser Phe Thr
Asp385 390 395 400Ala Ile
Phe Asp Glu Trp Met Lys Arg Phe Asn Pro Pro Ala Asp Ala
405 410 415Trp Pro Gln Glu Leu Ala Pro
Ile Gly His Asn Arg Met Tyr Asn Met 420 425
430Val Pro Phe Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu
Thr Ser 435 440 445Asp Gln Leu Gly
Tyr Ser Tyr Ala Ile Asp Leu Pro Val Ser Val Glu 450
455 460Glu Thr Pro Gly Trp Pro Thr Thr Leu Leu Val Val
Met Gly Thr Leu465 470 475
480Val Ala Leu Val Gly Leu Phe Val Leu Leu Ala Phe Leu Gln Tyr Arg
485 490 495Arg Leu Arg Lys Gly
Tyr Thr Pro Leu Met Glu Thr His Leu Ser Ser 500
505 510Lys Arg Tyr Thr Glu Glu Ala 5154439PRTHomo
sapiens 443Tyr Val Ile Thr Thr Gln His Trp Leu1
54449PRTHomo sapiens 444Leu Gly Tyr Ser Tyr Ala Ile Asp Leu1
544510PRTHomo sapiens 445Val Arg Ala Asp Thr Arg Pro Trp Ser Gly1
5 104469PRTHomo sapiens 446Leu Val Gly Leu Phe
Val Leu Leu Ala1 54479PRTHomo sapiens 447Leu Ile Ser Arg
Asn Ser Arg Phe Ser1 54489PRTHomo sapiens 448Phe Val Val
Leu His Ser Phe Thr Asp1 54499PRTHomo sapiens 449Trp His
Arg Tyr His Leu Leu Cys Leu1 54509PRTHomo sapiens 450Leu
Arg Lys Gly Tyr Thr Pro Leu Met1 54519PRTHomo sapiens
451Val Met Ser Leu His Asn Leu Val His1 54529PRTHomo
sapiens 452Leu Val Val Met Gly Thr Leu Val Ala1
545314PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/TRP-2 antigenic epitope hybrid peptide 453Leu Arg Met Lys Xaa
Tyr Val Ile Thr Thr Gln His Trp Leu1 5
1045414PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/TRP-2 antigenic epitope hybrid peptide 454Leu Arg Met Lys Xaa
Leu Gly Tyr Ser Tyr Ala Ile Asp Leu1 5
1045514PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/TRP-2 antigenic epitope hybrid peptide 455Leu Arg Met Lys Xaa
Val Arg Ala Asp Thr Arg Pro Ser Gly1 5
104569PRTHomo sapiens 456Ser Arg Phe Ser Ser Trp Glu Thr Val1
54579PRTHomo sapiens 457Lys Arg Phe Asn Pro Pro Ala Asp Ala1
54589PRTHomo sapiens 458Ile Arg Asp Cys Leu Ser Leu Gln Lys1
54599PRTHomo sapiens 459Lys Arg Val His Pro Asp Tyr Val Ile1
54609PRTHomo sapiens 460Asn Arg Met Tyr Asn Met Val Pro Phe1
54619PRTHomo sapiens 461Gly Leu Phe Val Leu Leu Ala Phe Leu1
54629PRTHomo sapiens 462Ser Val Tyr Asp Phe Phe Val Trp Leu1
54639PRTHomo sapiens 463Leu Ala Phe Leu Gln Tyr Arg Arg Leu1
54649PRTHomo sapiens 464Asn Met Val Pro Phe Phe Pro Pro Val1
54659PRTHomo sapiens 465Phe Val Trp Leu His Tyr Tyr Ser Val1
54669PRTHomo sapiens 466Ser Val Tyr Asp Phe Phe Val Trp Leu1
54679PRTHomo sapiens 467Gly Pro Gly Arg Pro Tyr Arg Ala Ile1
54689PRTHomo sapiens 468Ala Ala Arg Pro Asp Asp Pro Thr Leu1
54699PRTHomo sapiens 469Gly Phe Asp Lys Ala Asp Gly Thr
Leu1 54709PRTHomo sapiens 470Lys Arg Phe Asn Pro Pro Ala
Asp Ala1 54719PRTHomo sapiens 471His Tyr Tyr Ser Val Arg
Asp Thr Leu1 54729PRTHomo sapiens 472Leu Gln Lys Phe Asp
Asn Pro Pro Phe1 54739PRTHomo sapiens 473Ser Val Tyr Asp
Phe Phe Val Trp Leu1 54749PRTHomo sapiens 474Thr Leu Asp
Ser Gln Val Met Ser Leu1 54759PRTHomo sapiens 475Ser Leu
Asp Asp Tyr Asn His Leu Val1 54769PRTHomo sapiens 476Tyr
Ala Ile Asp Leu Pro Val Ser Val1 547723PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/TRP-2
hybrid peptide 477Leu Arg Met Lys Xaa Tyr Val Ile Thr Thr Gln His Trp Leu
Ser Val1 5 10 15Tyr Asp
Phe Phe Val Trp Leu 2047818PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/TRP-2 hybrid peptide 478Leu
Arg Met Lys Xaa Leu Gly Tyr Ser Tyr Ala Ile Asp Leu Pro Val1
5 10 15Ser Val47919PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/TRP-2
hybrid peptide 479Leu Arg Met Lys Xaa Thr Leu Asp Ser Gln Val Met Ser Leu
His Asn1 5 10 15Leu Val
His48010PRTHomo sapiens 480Cys Leu Leu Trp Ser Phe Gln Thr Ser Ala1
5 104819PRTHomo sapiens 481Ile Met Asp Gln Val
Pro Ser Phe Val1 54829PRTHomo sapiens 482Tyr Met Asp Gly
Thr Met Ser Gln Val1 548312PRTHomo sapiens 483Asp Ala Glu
Lys Ser Asp Ile Cys Thr Asp Glu Tyr1 5
104849PRTHomo sapiens 484Tyr Leu Glu Pro Gly Pro Val Thr Ala1
54859PRTHomo sapiens 485Tyr Met Asp Gly Thr Met Ser Gln Val1
54869PRTHomo sapiens 486Ala Leu Leu Ala Val Gly Ala Thr Lys1
5487529PRTHomo sapiens 487Met Leu Leu Ala Val Leu Tyr Cys Leu Leu Trp
Ser Phe Gln Thr Ser1 5 10
15Ala Gly His Phe Pro Arg Ala Cys Val Ser Ser Lys Asn Leu Met Glu
20 25 30Lys Glu Cys Cys Pro Pro Trp
Ser Gly Asp Arg Ser Pro Cys Gly Gln 35 40
45Leu Ser Gly Arg Gly Ser Cys Gln Asn Ile Leu Leu Ser Asn Ala
Pro 50 55 60Leu Gly Pro Gln Phe Pro
Phe Thr Gly Val Asp Asp Arg Glu Ser Trp65 70
75 80Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys
Ser Gly Asn Phe Met 85 90
95Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys
100 105 110Thr Glu Arg Arg Leu Leu
Val Arg Arg Asn Ile Phe Asp Leu Ser Ala 115 120
125Pro Glu Lys Asp Lys Phe Phe Ala Tyr Leu Thr Leu Ala Lys
His Thr 130 135 140Ile Ser Ser Asp Tyr
Val Ile Pro Ile Gly Thr Tyr Gly Gln Met Lys145 150
155 160Asn Gly Ser Thr Pro Met Phe Asn Asp Ile
Asn Ile Tyr Asp Leu Phe 165 170
175Val Trp Met His Tyr Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Ser
180 185 190Glu Ile Trp Arg Asp
Ile Asp Phe Ala His Glu Ala Pro Ala Phe Leu 195
200 205Pro Trp His Arg Leu Phe Leu Leu Arg Trp Glu Gln
Glu Ile Gln Lys 210 215 220Leu Thr Gly
Asp Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp225
230 235 240Ala Glu Lys Cys Asp Ile Cys
Thr Asp Glu Tyr Met Gly Gly Gln His 245
250 255Pro Thr Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe
Phe Ser Ser Trp 260 265 270Gln
Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu 275
280 285Cys Asn Gly Thr Pro Glu Gly Pro Leu
Arg Arg Asn Pro Gly Asn His 290 295
300Asp Lys Ser Arg Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe305
310 315 320Cys Leu Ser Leu
Thr Gln Tyr Glu Ser Gly Ser Met Asp Lys Ala Ala 325
330 335Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly
Phe Ala Ser Pro Leu Thr 340 345
350Gly Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile
355 360 365Tyr Met Asn Gly Thr Met Ser
Gln Val Gln Gly Ser Ala Asn Asp Pro 370 375
380Ile Phe Leu Leu His His Ala Phe Val Asp Ser Ile Phe Glu Gln
Trp385 390 395 400Leu Arg
Arg His Arg Pro Leu Gln Glu Val Tyr Pro Glu Ala Asn Ala
405 410 415Pro Ile Gly His Asn Arg Glu
Ser Tyr Met Val Pro Phe Ile Pro Leu 420 425
430Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly
Tyr Asp 435 440 445Tyr Ser Tyr Leu
Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp Tyr Ile 450
455 460Lys Ser Tyr Leu Glu Gln Ala Ser Arg Ile Trp Ser
Trp Leu Leu Gly465 470 475
480Ala Ala Met Val Gly Ala Val Leu Thr Ala Leu Leu Ala Gly Leu Val
485 490 495Ser Leu Leu Cys Arg
His Lys Arg Lys Gln Leu Pro Glu Glu Lys Gln 500
505 510Pro Leu Leu Met Glu Lys Glu Asp Tyr His Ser Leu
Tyr Gln Ser His 515 520 525Leu
4889PRTHomo sapiens 488Leu Arg Arg His Arg Pro Leu Gln Glu1
54899PRTHomo sapiens 489Met His Tyr Tyr Val Ser Met Asp Ala1
54909PRTHomo sapiens 490Trp Leu Arg Arg His Arg Pro Leu Gln1
54919PRTHomo sapiens 491Leu Val Arg Arg Asn Ile Phe Asp Leu1
54929PRTHomo sapiens 492Leu His Ile Tyr Met Asn Gly Thr Met1
54939PRTHomo sapiens 493Ile Tyr Met Asn Gly Thr Met Ser Gln1
54949PRTHomo sapiens 494Tyr Val Ser Met Asp Ala Leu Leu Gly1
54959PRTHomo sapiens 495Val Ile Pro Ile Gly Thr Tyr Gly Gln1
54969PRTHomo sapiens 496Phe Ser Phe Arg Asn Thr Leu Glu Gly1
54979PRTHomo sapiens 497Leu Leu Cys Arg His Lys Arg Lys Gln1
54989PRTHomo sapiens 498Met Leu Leu Ala Val Leu Tyr Cys Leu1
54999PRTHomo sapiens 499Phe Asn Asp Ile Asn Ile Tyr Asp Leu1
550015PRTHomo sapiens 500Gln Asn Ile Leu Leu Ser Asn Ala Pro
Leu Gly Pro Gln Phe Pro1 5 10
1550117PRTHomo sapiens 501Ala Leu His Ile Tyr Met Asn Gly Thr Met
Ser Gln Val Gln Gly Ser1 5 10
15Ala50220PRTHomo sapiens 502Tyr Gly Gln Met Lys Asn Gly Ser Thr Pro
Met Phe Asn Asp Ile Asn1 5 10
15Ile Tyr Asp Leu 2050320PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/tyrosinase non-overlapping hybrid peptide 503Leu Arg Met Lys Xaa
Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly1 5
10 15Pro Gln Phe Pro
2050414PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/tyrosinase non-overlapping hybrid peptide 504Leu Arg Met Lys
Xaa Leu Val Arg Arg Asn Ile Phe Asp Leu1 5
1050514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/tyrosinase non-overlapping hybrid peptide 505Leu
Arg Met Lys Xaa Phe Ser Phe Arg Asn Thr Leu Glu Gly1 5
1050614PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/tyrosinase non-overlapping hybrid peptide
506Leu Arg Met Lys Xaa Leu Leu Cys Arg His Lys Arg Lys Gln1
5 1050722PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/tyrosinase overlapping hybrid
peptide 507Leu Arg Met Lys Xaa Ala Leu His Ile Tyr Met Asn Gly Thr Met
Ser1 5 10 15Gln Val Gln
Gly Ser Ala 2050814PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/tyrosinase overlapping hybrid
peptide 508Leu Arg Met Lys Xaa Leu His Ile Tyr Met Asn Gly Thr Met1
5 1050914PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/tyrosinase overlapping hybrid
peptide 509Leu Arg Met Lys Xaa Ile Tyr Met Asn Gly Thr Met Ser Gln1
5 1051017PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/tyrosinase overlapping hybrid
peptide 510Leu Arg Met Lys Xaa Ala Leu His Ile Tyr Met Asn Gly Thr Met
Ser1 5 10
15Gln51114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/tyrosinase overlapping hybrid peptide 511Leu Arg
Met Lys Xaa Tyr Val Ser Met Asp Ala Leu Leu Gly1 5
1051214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/tyrosinase overlapping hybrid peptide 512Leu Arg
Met Lys Xaa Met His Tyr Tyr Val Ser Met Asp Ala1 5
1051317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/tyrosinase overlapping hybrid peptide 513Leu Arg
Met Lys Xaa Met His Tyr Tyr Val Ser Met Asp Ala Leu Leu1 5
10 15Gly51414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/tyrosinase overlapping hybrid peptide 514Leu Arg Met Lys Xaa Val
Ile Pro Ile Gly Thr Tyr Gly Gln1 5
1051525PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/tyrosinase overlapping hybrid peptide 515Leu Arg Met Lys Xaa
Tyr Gly Gln Met Lys Asn Gly Ser Thr Pro Met1 5
10 15Phe Asn Asp Ile Asn Ile Tyr Asp Leu
20 2551614PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/tyrosinase overlapping hybrid
peptide 516Leu Arg Met Lys Xaa Phe Asn Asp Ile Asn Ile Tyr Asp Leu1
5 1051731PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/tyrosinase overlapping hybrid
peptide 517Leu Arg Met Lys Xaa Val Ile Pro Ile Gly Thr Tyr Gly Gln Met
Lys1 5 10 15Asn Gly Ser
Thr Pro Met Phe Asn Asp Ile Asn Ile Tyr Asp Leu 20
25 305189PRTHomo sapiens 518Lys Cys Asp Ile Cys Thr
Asp Glu Tyr1 55199PRTHomo sapiens 519Tyr Met Asn Gly Thr
Met Ser Gln Val1 55209PRTHomo sapiens 520Met Leu Leu Ala
Val Leu Tyr Cys Leu1 55219PRTHomo sapiens 521Phe Leu Pro
Trp His Arg Leu Phe Leu1 552212PRTHomo sapiens 522Asp Ala
Glu Lys Ser Asp Ile Cys Thr Asp Glu Tyr1 5
105239PRTHomo sapiens 523Tyr Met Asp Gly Thr Met Ser Gln Val1
552411PRTHomo sapiens 524Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr1
5 1052532PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/tyrosinase non-overlapping
hybrid peptide 525Leu Arg Met Lys Xaa Asp Ala Glu Lys Ser Asp Ile Cys Thr
Asp Glu1 5 10 15Tyr Gln
Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro 20
25 3052623PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/tyrosinase non-overlapping
hybrid peptide 526Leu Arg Met Lys Xaa Phe Leu Pro Trp His Arg Leu Phe Leu
Leu Arg1 5 10 15Arg His
Arg Pro Leu Gln Glu 2052722PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/tyrosinase overlapping
hybrid peptide 527Leu Arg Met Lys Xaa Ala Leu His Ile Tyr Met Asn Gly Thr
Met Ser1 5 10 15Gln Val
Gln Gly Ser Ala 2052835PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/tyrosinase overlapping hybrid
peptide 528Leu Arg Met Lys Xaa Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr
Tyr1 5 10 15Gly Gln Met
Lys Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile 20
25 30Tyr Asp Leu 3552914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/tyrosinase overlapping hybrid peptide 529Leu Arg Met Lys Xaa Met
Leu Leu Ala Val Leu Tyr Cys Leu1 5
105309PRTHomo sapiens 530Ala Ala Gly Ile Gly Ile Leu Thr Val1
5531118PRTHomo sapiens 531Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly
Tyr Pro Lys Lys Gly1 5 10
15His Gly His Ser Tyr Thr Thr Ala Glu Glu Ala Ala Gly Ile Gly Ile
20 25 30Leu Thr Val Ile Leu Gly Val
Leu Leu Leu Ile Gly Cys Trp Tyr Cys 35 40
45Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His
Val 50 55 60Gly Thr Gln Cys Ala Leu
Thr Arg Arg Cys Pro Gln Glu Gly Phe Asp65 70
75 80His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys
Asn Cys Glu Pro Val 85 90
95Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser
100 105 110Pro Pro Pro Tyr Ser Pro
1155329PRTHomo sapiens 532Leu Thr Val Ile Leu Gly Val Leu Leu1
55339PRTHomo sapiens 533Val Ile Leu Gly Val Leu Leu Leu Ile1
55349PRTHomo sapiens 534Val Val Pro Asn Ala Pro Pro Ala Tyr1
55359PRTHomo sapiens 535Ile Gly Ile Leu Thr Val Ile Leu Gly1
55369PRTHomo sapiens 536Ile Tyr Gly Tyr Pro Lys Lys Gly His1
553723PRTHomo sapiens 537Arg Asn Gly Tyr Arg Ala Leu Met Asp
Lys Ser Leu His Val Gly Thr1 5 10
15Gln Cys Ala Leu Thr Arg Arg 2053814PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/MART-1/Melan-1 non-overlapping hybrid peptide 538Leu Arg Met
Lys Xaa Val Val Pro Asn Ala Pro Pro Ala Tyr1 5
1053914PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MART-1/Melan-1 non-overlapping hybrid peptide
539Leu Arg Met Lys Xaa Ile Tyr Gly Tyr Pro Lys Lys Gly His1
5 1054028PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/MART-1/Melan-1 non-overlapping
hybrid peptide 540Leu Arg Met Lys Xaa Arg Asn Gly Tyr Arg Ala Leu Met
Asp Lys Ser1 5 10 15Leu
His Val Gly Thr Gln Cys Ala Leu Thr Arg Arg 20
2554114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MART-1/Melan-1 overlapping hybrid peptide
541Leu Arg Met Lys Xaa Leu Thr Val Ile Leu Gly Val Leu Leu1
5 1054214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/MART-1/Melan-1 overlapping
hybrid peptide 542Leu Arg Met Lys Xaa Val Ile Leu Gly Val Leu Leu Leu
Ile1 5 1054314PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/MART-1/Melan-1 overlapping hybrid peptide 543Leu Arg Met Lys
Xaa Ile Gly Ile Leu Thr Val Ile Leu Gly1 5
1054419PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MART-1/Melan-1 overlapping hybrid peptide
544Leu Arg Met Lys Xaa Ile Gly Ile Leu Thr Val Ile Leu Gly Val Leu1
5 10 15Leu Leu Ile5459PRTHomo
sapiens 545Leu Leu Leu Ile Gly Cys Trp Tyr Cys1
55469PRTHomo sapiens 546Ala Leu Met Asp Lys Ser Leu His Val1
55479PRTHomo sapiens 547Glu Glu Ala Ala Gly Ile Gly Ile Leu1
55489PRTHomo sapiens 548Ala Glu Gln Ser Pro Pro Pro Tyr Ser1
55499PRTHomo sapiens 549Ala Ala Gly Ile Gly Ile Leu Thr Val1
55509PRTHomo sapiens 550Ile Leu Thr Val Ile Leu Gly Val Leu1
555111PRTHomo sapiens 551Ala Glu Glu Ala Ala Gly Ile Gly Ile Leu
Thr1 5 1055237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/MART-1
hybrid peptide 552Leu Arg Met Lys Xaa Ala Ala Gly Ile Gly Ile Leu Thr Val
Arg Asn1 5 10 15Gly Tyr
Arg Ala Leu Met Asp Lys Ser Leu His Val Gly Thr Gln Cys 20
25 30Ala Leu Thr Arg Arg
355531255PRTHomo sapiens 553Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu
Leu Leu Ala Leu Leu1 5 10
15Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys
20 25 30Leu Arg Leu Pro Ala Ser Pro
Glu Thr His Leu Asp Met Leu Arg His 35 40
45Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr
Tyr 50 55 60Leu Pro Thr Asn Ala Ser
Leu Ser Phe Leu Gln Asp Ile Gln Glu Val65 70
75 80Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val
Arg Gln Val Pro Leu 85 90
95Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr
100 105 110Ala Leu Ala Val Leu Asp
Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120
125Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu
Arg Ser 130 135 140Leu Thr Glu Ile Leu
Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln145 150
155 160Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys
Asp Ile Phe His Lys Asn 165 170
175Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys
180 185 190His Pro Cys Ser Pro
Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195
200 205Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys
Ala Gly Gly Cys 210 215 220Ala Arg Cys
Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys225
230 235 240Ala Ala Gly Cys Thr Gly Pro
Lys His Ser Asp Cys Leu Ala Cys Leu 245
250 255His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys
Pro Ala Leu Val 260 265 270Thr
Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg 275
280 285Tyr Thr Phe Gly Ala Ser Cys Val Thr
Ala Cys Pro Tyr Asn Tyr Leu 290 295
300Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln305
310 315 320Glu Val Thr Ala
Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325
330 335Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly
Met Glu His Leu Arg Glu 340 345
350Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys
355 360 365Lys Ile Phe Gly Ser Leu Ala
Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375
380Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val
Phe385 390 395 400Glu Thr
Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro
405 410 415Asp Ser Leu Pro Asp Leu Ser
Val Phe Gln Asn Leu Gln Val Ile Arg 420 425
430Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln
Gly Leu 435 440 445Gly Ile Ser Trp
Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450
455 460Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe
Val His Thr Val465 470 475
480Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr
485 490 495Ala Asn Arg Pro Glu
Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500
505 510Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly
Pro Thr Gln Cys 515 520 525Val Asn
Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530
535 540Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val
Asn Ala Arg His Cys545 550 555
560Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys
565 570 575Phe Gly Pro Glu
Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580
585 590Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly
Val Lys Pro Asp Leu 595 600 605Ser
Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610
615 620Pro Cys Pro Ile Asn Cys Thr His Ser Cys
Val Asp Leu Asp Asp Lys625 630 635
640Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile
Ser 645 650 655Ala Val Val
Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly 660
665 670Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile
Arg Lys Tyr Thr Met Arg 675 680
685Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690
695 700Ala Met Pro Asn Gln Ala Gln Met
Arg Ile Leu Lys Glu Thr Glu Leu705 710
715 720Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly
Thr Val Tyr Lys 725 730
735Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile
740 745 750Lys Val Leu Arg Glu Asn
Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 755 760
765Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val
Ser Arg 770 775 780Leu Leu Gly Ile Cys
Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu785 790
795 800Met Pro Tyr Gly Cys Leu Leu Asp His Val
Arg Glu Asn Arg Gly Arg 805 810
815Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly
820 825 830Met Ser Tyr Leu Glu
Asp Val Arg Leu Val His Arg Asp Leu Ala Ala 835
840 845Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys
Ile Thr Asp Phe 850 855 860Gly Leu Ala
Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp865
870 875 880Gly Gly Lys Val Pro Ile Lys
Trp Met Ala Leu Glu Ser Ile Leu Arg 885
890 895Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr
Gly Val Thr Val 900 905 910Trp
Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915
920 925Arg Glu Ile Pro Asp Leu Leu Glu Lys
Gly Glu Arg Leu Pro Gln Pro 930 935
940Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met945
950 955 960Ile Asp Ser Glu
Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe 965
970 975Ser Arg Met Ala Arg Asp Pro Gln Arg Phe
Val Val Ile Gln Asn Glu 980 985
990Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu
995 1000 1005Leu Glu Asp Asp Asp Met Gly
Asp Leu Val Asp Ala Glu Glu Tyr Leu 1010 1015
1020Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala
Gly1025 1030 1035 1040Gly Met
Val His His Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly
1045 1050 1055Gly Asp Leu Thr Leu Gly Leu
Glu Pro Ser Glu Glu Glu Ala Pro Arg 1060 1065
1070Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp Val Phe
Asp Gly 1075 1080 1085Asp Leu Gly
Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His 1090
1095 1100Asp Pro Ser Pro Leu Gln Arg Tyr Ser Glu Asp Pro
Thr Val Pro Leu1105 1110 1115
1120Pro Ser Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln
1125 1130 1135Pro Glu Tyr Val Asn
Gln Pro Asp Val Arg Pro Gln Pro Pro Ser Pro 1140
1145 1150Arg Glu Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly
Ala Thr Leu Glu 1155 1160 1165Arg
Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val 1170
1175 1180Phe Ala Phe Gly Gly Ala Val Glu Asn Pro
Glu Tyr Leu Thr Pro Gln1185 1190 1195
1200Gly Gly Ala Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro
Ala 1205 1210 1215Phe Asp
Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala 1220
1225 1230Pro Pro Ser Thr Phe Lys Gly Thr Pro
Thr Ala Glu Asn Pro Glu Tyr 1235 1240
1245Leu Gly Leu Asp Val Pro Val 1250 12555549PRTHomo
sapiens 554Phe Val Val Ile Gln Asn Glu Asp Leu1
55559PRTHomo sapiens 555Leu Arg Ile Val Arg Gly Thr Gln Leu1
55569PRTHomo sapiens 556Met Ile Met Val Lys Cys Trp Met Ile1
55579PRTHomo sapiens 557Leu Arg Arg Arg Phe Thr His Gln Ser1
55589PRTHomo sapiens 558Tyr Thr Met Arg Arg Leu Leu Gln Glu1
55599PRTHomo sapiens 559Val Val Leu Gly Val Val Phe Gly Ile1
55609PRTHomo sapiens 560Met Val His His Arg His Arg Ser Ser1
55619PRTHomo sapiens 561Leu Ser Val Phe Gln Asn Leu Gln Val1
55629PRTHomo sapiens 562Leu Thr Leu Ile Asp Thr Asn Arg Ser1
55639PRTHomo sapiens 563Phe Gly Ile Leu Ile Lys Arg Arg Gln1
55649PRTHomo sapiens 564Leu Gln Val Phe Glu Thr Leu Glu Glu1
55659PRTHomo sapiens 565Leu Thr Tyr Leu Pro Thr Asn Ala Ser1
55669PRTHomo sapiens 566Tyr Met Ile Met Val Lys Cys Trp Met1
55679PRTHomo sapiens 567Leu Arg Lys Val Lys Val Leu Gly Ser1
55689PRTHomo sapiens 568Phe Gln Asn Leu Gln Val Ile Arg Gly1
556915PRTHomo sapiens 569Val Pro Ile Lys Trp Met Ala Leu
Glu Ser Ile Leu Arg Arg Arg1 5 10
1557013PRTHomo sapiens 570Gly Ser Pro Tyr Val Ser Arg Leu Leu
Gly Ile Cys Leu1 5 1057111PRTHomo sapiens
571Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile1 5
1057213PRTHomo sapiens 572Leu Cys Phe Val His Thr Val Pro Trp Asp
Gln Leu Phe1 5 1057312PRTHomo sapiens
573Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu1 5
1057412PRTHomo sapiens 574Glu Phe Ser Arg Met Ala Arg Asp Pro
Gln Arg Phe1 5 1057512PRTHomo sapiens
575Phe Asp Gly Asp Leu Gly Met Ala Ala Lys Gly Leu1 5
1057615PRTHomo sapiens 576His Leu Asp Met Leu Arg His Leu Tyr
Gln Gly Cys Gln Val Val1 5 10
1557716PRTHomo sapiens 577Leu Arg Ile Val Arg Gly Thr Gln Leu Phe
Glu Asp Asn Tyr Ala Leu1 5 10
1557818PRTHomo sapiens 578Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro
Cys Ala Arg Val Cys Tyr1 5 10
15Gly Leu57915PRTHomo sapiens 579Leu Gly Ser Gly Ala Phe Gly Thr Val
Tyr Lys Gly Ile Trp Ile1 5 10
1558015PRTHomo sapiens 580Pro Ala Arg Glu Ile Pro Asp Leu Leu Glu
Lys Gly Glu Arg Leu1 5 10
1558115PRTHomo sapiens 581Thr Leu Glu Arg Pro Lys Thr Leu Ser Pro Gly
Lys Asn Gly Val1 5 10
1558217PRTHomo sapiens 582Lys Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu
Ser Phe Asp Gly1 5 10
15Asp58316PRTHomo sapiens 583Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg
Arg Leu Leu Gln Glu1 5 10
1558414PRTHomo sapiens 584Glu Leu Val Ser Glu Phe Ser Arg Met Ala Arg
Asp Pro Gln1 5 1058510PRTHomo sapiens
585Leu Arg Ile Val Arg Thr Gly Thr Gln Leu1 5
105869PRTHomo sapiens 586Leu Val Ser Glu Phe Ser Arg Met Ala1
558718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Her-2/neu non-overlapping hybrid peptide 587Leu Arg
Met Lys Xaa Gly Ser Pro Tyr Val Ser Arg Leu Leu Gly Ile1 5
10 15Cys Leu58816PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/Her-2/neu non-overlapping hybrid peptide 588Leu Arg Met Lys Xaa
Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile1 5
10 1558914PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Her-2/neu non-overlapping
hybrid peptide 589Leu Arg Met Lys Xaa Phe Val Val Ile Gln Asn Glu Asp
Leu1 5 1059014PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/Her-2/neu non-overlapping hybrid peptide 590Leu Arg Met Lys Xaa
Leu Arg Ile Val Arg Gly Thr Gln Leu1 5
1059114PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Her-2/neu non-overlapping hybrid peptide 591Leu Arg Met Lys
Xaa Leu Arg Arg Arg Phe Thr His Gln Ser1 5
1059214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Her-2/neu non-overlapping hybrid peptide 592Leu Arg
Met Lys Xaa Tyr Thr Met Arg Arg Leu Leu Gln Glu1 5
1059314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Her-2/neu non-overlapping hybrid peptide 593Leu Arg
Met Lys Xaa Met Val His His Arg His Arg Ser Ser1 5
1059414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Her-2/neu non-overlapping hybrid peptide 594Leu Arg
Met Lys Xaa Leu Val Ser Glu Phe Ser Arg Met Ala1 5
1059525PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Her-2/neu overlapping hybrid peptide 595Leu Arg Met
Lys Xaa Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile1 5
10 15Leu Arg Arg Arg Phe Thr His Gln Ser
20 2559620PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Her-2/neu overlapping hybrid
peptide 596Leu Arg Met Lys Xaa Val Val Leu Gly Val Val Phe Gly Ile Leu
Ile1 5 10 15Lys Arg Arg
Gln 2059715PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Her-2/neu overlapping hybrid peptide
597Leu Arg Met Lys Xaa Tyr Met Ile Met Val Lys Cys Trp Met Ile1
5 10 1559817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/Her-2/neu overlapping hybrid peptide 598Leu Arg Met Lys Xaa Leu
Ser Val Phe Gln Asn Leu Gln Val Ile Arg1 5
10 15Gly5999PRTHomo sapiens 599Ile Leu Leu Val Val Val
Leu Gly Val1 56009PRTHomo sapiens 600Lys Ile Phe Gly Ser
Leu Ala Phe Leu1 56019PRTHomo sapiens 601Ile Leu Trp Lys
Asp Ile Phe His Lys1 56029PRTHomo sapiens 602Thr Tyr Leu
Pro Thr Asn Ala Ser Leu1 56039PRTHomo sapiens 603Gln Leu
Phe Glu Asp Asn Tyr Ala Leu1 56049PRTHomo sapiens 604Glu
Tyr Val Asn Ala Arg His Cys Leu1 56059PRTHomo sapiens
605Ala Tyr Ser Leu Thr Leu Gln Gly Leu1 56069PRTHomo
sapiens 606Ser Tyr Gly Val Thr Val Trp Glu Leu1
56079PRTHomo sapiens 607Glu Tyr Leu Val Pro Gln Gln Gly Phe1
56089PRTHomo sapiens 608Arg Leu Leu Gln Glu Thr Glu Leu Val1
56099PRTHomo sapiens 609Ile Leu Lys Glu Thr Glu Leu Arg Lys1
56109PRTHomo sapiens 610Val Leu Arg Glu Asn Thr Ser Pro Lys1
56119PRTHomo sapiens 611Ile Leu Ile Lys Arg Arg Gln Gln Lys1
56129PRTHomo sapiens 612Gln Leu Phe Glu Asp Asn Tyr Ala Leu1
56139PRTHomo sapiens 613Lys Ile Phe Gly Ser Leu Ala Phe Leu1
56149PRTHomo sapiens 614Arg Leu Leu Gln Glu Thr Glu Leu Val1
56159PRTHomo sapiens 615Ile Leu His Asn Gly Ala Tyr Ser Leu1
56169PRTHomo sapiens 616Val Val Leu Gly Val Val Phe Gly Ile1
56179PRTHomo sapiens 617Tyr Met Ile Met Val Lys Cys Trp Met1
56189PRTHomo sapiens 618Ile Ile Ser Ala Val Val Gly Ile Leu1
561910PRTHomo sapiens 619Phe Leu Ser Ala Val Val Gly Ile Leu
Val1 5 1062010PRTHomo sapiens 620Val Met
Ala Gly Val Gly Ser Pro Tyr Val1 5
106219PRTHomo sapiens 621Val Leu Arg Glu Asn Thr Ser Pro Lys1
562223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MHC Class II epitope/MHC Class I epitope
hybrid peptide 622Leu Arg Met Lys Xaa Val Met Ala Gly Val Gly Ser Pro Tyr
Val Ser1 5 10 15Arg Leu
Leu Gly Ile Cys Leu 2062325PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/MHC Class II epitope/MHC
Class I epitope hybrid peptide 623Leu Arg Met Lys Xaa Gln Leu Gln
Val Phe Glu Thr Leu Glu Glu Ile1 5 10
15Lys Ile Phe Gly Ser Leu Ala Phe Leu 20
2562416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MHC Class II epitope/MHC Class I epitope
hybrid peptide 624Leu Arg Met Lys Xaa Phe Gly Ile Leu Ile Lys Arg Arg Gln
Gln Lys1 5 10
1562515PRTHomo sapiens 625Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys
Lys Gly Pro1 5 10
1562615PRTHomo sapiens 626Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu
Pro Thr Asp1 5 10
1562715PRTHomo sapiens 627Gln Phe Leu Arg Gln Glu Cys Val Glu Glu Cys Arg
Val Leu Gln1 5 10
1562815PRTHomo sapiens 628Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala
Arg His Cys1 5 10
1562915PRTHomo sapiens 629Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp
Gln Cys Val1 5 10
1563037PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Her-2/neu non-overlapping hybrid peptide 630Leu Arg Met Lys
Xaa Gly Ser Pro Tyr Val Ser Arg Leu Leu Gly Ile1 5
10 15Cys Leu Thr Arg Thr Val Cys Ala Gly Gly
Cys Ala Arg Cys Lys Gly 20 25
30Pro Leu Pro Thr Asp 3563131PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Her-2/neu non-overlapping
hybrid peptide 631Leu Arg Met Lys Xaa Gln Leu Gln Val Phe Glu Thr Leu Glu
Glu Ile1 5 10 15Asn Gly
Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val 20
25 3063222PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Her-2/neu overlapping hybrid
peptide 632Leu Arg Met Lys Xaa Ser Gln Phe Leu Arg Gly Gln Glu Cys Val
Glu1 5 10 15Glu Cys Arg
Val Leu Gln 2063321PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Her-2/neu overlapping hybrid
peptide 633Leu Arg Met Lys Xaa Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr
Val1 5 10 15Asn Ala Arg
His Cys 2063414PRTArtificial SequenceDescription of Artificial
Sequence Synthetic MHC Class II-presented LF epitope peptide 634His
Ile Ser Leu Glu Ala Leu Ser Asp Lys Lys Lys Ile Lys1 5
1063515PRTArtificial SequenceDescription of Artificial
Sequence Synthetic MHC Class II-presented LF epitope peptide 635Glu
Gln Glu Ile Asn Leu Ser Leu Glu Glu Leu Lys Asp Gln Arg1 5
10 1563615PRTArtificial
SequenceDescription of Artificial Sequence Synthetic MHC Class
II-presented LF epitope peptide 636Asp Asp Ile Ile His Ser Leu Ser Gln
Glu Glu Lys Glu Leu Leu1 5 10
15637776PRTBacillus anthracis 637Ala Gly Gly His Gly Asp Val Gly Met
His Val Lys Glu Lys Glu Lys1 5 10
15Asn Lys Asp Glu Asn Lys Arg Lys Asp Glu Glu Arg Asn Lys Thr
Gln 20 25 30Glu Glu His Leu
Lys Glu Ile Met Lys His Ile Val Lys Ile Glu Val 35
40 45Lys Gly Glu Glu Ala Val Lys Lys Glu Ala Ala Glu
Lys Leu Leu Glu 50 55 60Lys Val Pro
Ser Asp Val Leu Glu Met Tyr Lys Ala Ile Gly Gly Lys65 70
75 80Ile Tyr Ile Val Asp Gly Asp Ile
Thr Lys His Ile Ser Leu Glu Ala 85 90
95Leu Ser Glu Asp Lys Lys Lys Ile Lys Asp Ile Tyr Gly Lys
Asp Ala 100 105 110Leu Leu His
Glu His Tyr Val Tyr Ala Lys Glu Gly Tyr Glu Pro Val 115
120 125Leu Val Ile Gln Ser Ser Glu Asp Tyr Val Glu
Asn Thr Glu Lys Ala 130 135 140Leu Asn
Val Tyr Tyr Glu Ile Gly Lys Ile Leu Ser Arg Asp Ile Leu145
150 155 160Ser Lys Ile Asn Gln Pro Tyr
Gln Lys Phe Leu Asp Val Leu Asn Thr 165
170 175Ile Lys Asn Ala Ser Asp Ser Asp Gly Gln Asp Leu
Leu Phe Thr Asn 180 185 190Gln
Leu Lys Glu His Pro Thr Asp Phe Ser Val Glu Phe Leu Glu Gln 195
200 205Asn Ser Asn Glu Val Gln Glu Val Phe
Ala Lys Ala Phe Ala Tyr Tyr 210 215
220Ile Glu Pro Gln His Arg Asp Val Leu Gln Leu Tyr Ala Pro Glu Ala225
230 235 240Phe Asn Tyr Met
Asp Lys Phe Asn Glu Gln Glu Ile Asn Leu Ser Leu 245
250 255Glu Glu Leu Lys Asp Gln Arg Met Leu Ser
Arg Tyr Glu Lys Trp Glu 260 265
270Lys Ile Lys Gln His Tyr Gln His Trp Ser Asp Ser Leu Ser Glu Glu
275 280 285Gly Arg Gly Leu Leu Lys Lys
Leu Gln Ile Pro Ile Glu Pro Lys Lys 290 295
300Asp Asp Ile Ile His Ser Leu Ser Gln Glu Glu Lys Glu Leu Leu
Lys305 310 315 320Arg Ile
Gln Ile Asp Ser Ser Asp Phe Leu Ser Thr Glu Glu Lys Glu
325 330 335Phe Leu Lys Lys Leu Gln Ile
Asp Ile Arg Asp Ser Leu Ser Glu Glu 340 345
350Glu Lys Glu Leu Leu Asn Arg Ile Gln Val Asp Ser Ser Asn
Pro Leu 355 360 365Ser Glu Lys Glu
Lys Glu Phe Leu Lys Lys Leu Lys Leu Asp Ile Gln 370
375 380Pro Tyr Asp Ile Asn Gln Arg Leu Gln Asp Thr Gly
Gly Leu Ile Asp385 390 395
400Ser Pro Ser Ile Asn Leu Asp Val Arg Lys Gln Tyr Lys Arg Asp Ile
405 410 415Gln Asn Ile Asp Ala
Leu Leu His Gln Ser Ile Gly Ser Thr Leu Tyr 420
425 430Asn Lys Ile Tyr Leu Tyr Glu Asn Met Asn Ile Asn
Asn Leu Thr Ala 435 440 445Thr Leu
Gly Ala Asp Leu Val Asp Ser Thr Asp Asn Thr Lys Ile Asn 450
455 460Arg Gly Ile Phe Asn Glu Phe Lys Lys Asn Phe
Lys Tyr Ser Ile Ser465 470 475
480Ser Asn Tyr Met Ile Val Asp Ile Asn Glu Arg Pro Ala Leu Asp Asn
485 490 495Glu Arg Leu Lys
Trp Arg Ile Gln Leu Ser Pro Asp Thr Arg Ala Gly 500
505 510Tyr Leu Glu Asn Gly Lys Leu Ile Leu Gln Arg
Asn Ile Gly Leu Glu 515 520 525Ile
Lys Asp Val Gln Ile Ile Lys Gln Ser Glu Lys Glu Tyr Ile Arg 530
535 540Ile Asp Ala Lys Val Val Pro Lys Ser Lys
Ile Asp Thr Lys Ile Gln545 550 555
560Glu Ala Gln Leu Asn Ile Asn Gln Glu Trp Asn Lys Ala Leu Gly
Leu 565 570 575Pro Lys Tyr
Thr Lys Leu Ile Thr Phe Asn Val His Asn Arg Tyr Ala 580
585 590Ser Asn Ile Val Glu Ser Ala Tyr Leu Ile
Leu Asn Glu Trp Lys Asn 595 600
605Asn Ile Gln Ser Asp Leu Ile Lys Lys Val Thr Asn Tyr Leu Val Asp 610
615 620Gly Asn Gly Arg Phe Val Phe Thr
Asp Ile Thr Leu Pro Asn Ile Ala625 630
635 640Glu Gln Tyr Thr His Gln Asp Glu Ile Tyr Glu Gln
Val His Ser Lys 645 650
655Gly Leu Tyr Val Pro Glu Ser Arg Ser Ile Leu Leu His Gly Pro Ser
660 665 670Lys Gly Val Glu Leu Arg
Asn Asp Ser Glu Gly Phe Ile His Glu Phe 675 680
685Gly His Ala Val Asp Asp Tyr Ala Gly Tyr Leu Leu Asp Lys
Asn Gln 690 695 700Ser Asp Leu Val Thr
Asn Ser Lys Lys Phe Ile Asp Ile Phe Lys Glu705 710
715 720Glu Gly Ser Asn Leu Thr Ser Tyr Gly Arg
Thr Asn Glu Ala Glu Phe 725 730
735Phe Ala Glu Ala Phe Arg Leu Met His Ser Thr Asp His Ala Glu Arg
740 745 750Leu Lys Val Gln Lys
Asn Ala Pro Lys Thr Phe Gln Phe Ile Asn Asp 755
760 765Gln Ile Lys Phe Ile Ile Asn Ser 770
7756389PRTBacillus anthracis 638Trp Arg Ile Gln Leu Ser Pro Asp Thr1
56399PRTBacillus anthracis 639Tyr Ile Arg Ile Asp Ala Lys
Val Val1 56409PRTBacillus anthracis 640Phe Arg Leu Met His
Ser Thr Asp His1 56419PRTBacillus anthracis 641Leu Gln Arg
Asn Ile Gly Leu Glu Ile1 56429PRTBacillus anthracis 642Leu
Gln Ile Asp Ile Arg Asp Ser Leu1 56439PRTBacillus anthracis
643Ile Asn Leu Asp Val Arg Lys Gln Tyr1 56449PRTBacillus
anthracis 644Leu Arg Asn Asp Ser Glu Gly Phe Ile1
56459PRTBacillus anthracis 645Leu Val Ile Gln Ser Ser Glu Asp Tyr1
56469PRTBacillus anthracis 646Tyr Leu Leu Asp Lys Asn Gln Ser Asp1
56479PRTBacillus anthracis 647Tyr Ser Ile Ser Ser Asn Tyr
Met Ile1 56489PRTBacillus anthracis 648Leu Ile Asp Ser Pro
Ser Ile Asn Leu1 56499PRTBacillus anthracis 649Ile Val Glu
Ser Ala Tyr Leu Ile Leu1 56509PRTBacillus anthracis 650Phe
Lys Tyr Ser Ile Ser Ser Asn Tyr1 56519PRTBacillus anthracis
651Phe Asn Tyr Met Asp Lys Phe Asn Glu1 56529PRTBacillus
anthracis 652Phe Leu Lys Lys Leu Lys Leu Asp Ile1
56539PRTBacillus anthracis 653Val Val Pro Lys Ser Lys Ile Asp Thr1
56549PRTBacillus anthracis 654Tyr Tyr Glu Ile Gly Lys Ile Leu Ser1
56559PRTBacillus anthracis 655Ile Gln Asn Ile Asp Ala Leu
Leu His1 56569PRTBacillus anthracis 656Leu Val Thr Asn Ser
Lys Lys Phe Ile1 56579PRTBacillus anthracis 657Leu Ile Thr
Phe Asn Val His Asn Arg1 56589PRTBacillus anthracis 658Leu
Glu Ile Lys Asp Val Gln Ile Ile1 56599PRTBacillus anthracis
659Ile Tyr Leu Tyr Glu Asn Met Asn Ile1 56609PRTBacillus
anthracis 660Leu Glu Met Tyr Lys Ala Ile Gly Gly1
56619PRTBacillus anthracis 661Phe Ile His Glu Phe Gly His Ala Val1
56629PRTBacillus anthracis 662Phe Ile Asn Asp Gln Ile Lys Phe Ile1
56639PRTBacillus anthracis 663Val Tyr Tyr Glu Ile Gly Lys
Ile Leu1 56649PRTBacillus anthracis 664His Tyr Gln His Trp
Ser Asp Ser Leu1 56659PRTBacillus anthracis 665Leu Leu His
Glu His Tyr Val Tyr Ala1 56669PRTBacillus anthracis 666Ser
Thr Glu Glu Lys Glu Phe Leu Lys1 56679PRTBacillus anthracis
667Lys Leu Gln Ile Pro Ile Glu Pro Lys1 56689PRTBacillus
anthracis 668Tyr Val Pro Glu Ser Arg Ser Ile Leu1
566913PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/MHC Class II epitope hybrid peptide 669Leu Arg Met Lys Trp Arg
Ile Gln Leu Ser Pro Asp Thr1 5
1067013PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/MHC Class II epitope hybrid peptide 670Leu Arg Met Lys Tyr
Ile Arg Ile Asp Ala Lys Val Val1 5
1067113PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/MHC Class II epitope hybrid peptide 671Leu Arg Met Lys Phe
Arg Leu Met His Ser Thr Asp His1 5
1067214PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/MHC Class II epitope hybrid peptide 672Leu Arg Met Lys Leu
Ile Gln Arg Asn Ile Gly Leu Glu Ile1 5
1067313PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/MHC Class II epitope hybrid peptide 673Leu Arg Met Lys Leu
Gln Ile Asp Ile Arg Asp Ser Leu1 5
1067417PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/MHC Class II epitope hybrid peptide 674Leu Arg Met Lys Ile
Asn Leu Asp Val Arg Lys Gln Tyr Lys Arg Asp1 5
10 15Ile67513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/MHC Class II epitope hybrid
peptide 675Leu Arg Met Lys Leu Arg Asn Asp Ser Glu Gly Phe Ile1
5 1067614PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/MHC Class II epitope hybrid
peptide 676Leu Arg Met Lys Tyr Glu Pro Val Gln Ser Ser Glu Asp Tyr1
5 1067723PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/anthrax lethal factor MHC Class
II epitope/ARD hybrid peptide 677Leu Arg Met Lys Pro Tyr Gln Lys Phe
Leu Asp Val Leu Asn Thr Ile1 5 10
15Lys Asn Ala Ser Asp Ser Asp 2067828PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/anthrax
lethal factor MHC Class II epitope/ARD hybrid peptide 678Leu Arg Met
Lys Thr Asn Gln Leu Lys Glu His Pro Thr Asp Phe Ser1 5
10 15Val Glu Phe Leu Glu Gln Asn Ser Asn
Glu Val Gln 20 2567930PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/anthrax
lethal factor MHC Class II epitope/ARD hybrid peptide 679Leu Arg Met
Lys Asp Phe Ser Val Glu Phe Leu Glu Gln Asn Ser Asn1 5
10 15Glu Val Gln Glu Val Phe Ala Lys Ala
Phe Ala Tyr Tyr Ile 20 25
3068019PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/anthrax lethal factor MHC Class II epitope/ARD hybrid
peptide 680Leu Arg Met Lys Gln His Arg Asp Val Leu Gln Leu Tyr Ala Pro
Glu1 5 10 15Ala Phe
Asn681764PRTBacillus anthracis 681Met Lys Lys Arg Lys Val Leu Ile Pro Leu
Met Ala Leu Ser Thr Ile1 5 10
15Leu Val Ser Ser Thr Gly Asn Leu Glu Val Ile Gln Ala Glu Val Lys
20 25 30Gln Glu Asn Arg Leu Leu
Asn Glu Ser Glu Ser Ser Ser Gln Gly Leu 35 40
45Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro Met
Val Val 50 55 60Thr Ser Ser Thr Thr
Gly Asp Leu Ser Ile Pro Ser Ser Glu Leu Glu65 70
75 80Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln
Ser Ala Ile Trp Ser Gly 85 90
95Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala Thr Ser Ala
100 105 110Asp Asn His Val Thr
Met Trp Val Asp Asp Gln Glu Val Ile Asn Lys 115
120 125Ala Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly
Arg Leu Tyr Gln 130 135 140Ile Lys Ile
Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys Gly Leu Asp145
150 155 160Phe Lys Leu Tyr Trp Thr Asp
Ser Gln Asn Lys Lys Glu Val Ile Ser 165
170 175Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys
Ser Ser Asn Ser 180 185 190Arg
Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro Asp Arg Asp 195
200 205Asn Asp Gly Ile Pro Asp Ser Leu Glu
Val Glu Gly Tyr Thr Val Asp 210 215
220Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser Asn Ile His225
230 235 240Glu Lys Lys Gly
Leu Thr Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser 245
250 255Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu
Lys Val Thr Gly Arg Ile 260 265
270Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val Ala Ala Tyr
275 280 285Pro Ile Val His Val Asp Met
Glu Asn Ile Ile Leu Ser Lys Asn Glu 290 295
300Asp Gln Ser Thr Gln Asn Thr Asp Ser Gln Thr Arg Thr Ile Ser
Lys305 310 315 320Asn Thr
Ser Thr Ser Arg Thr His Thr Ser Glu Val His Gly Asn Ala
325 330 335Glu Val His Ala Ser Phe Phe
Asp Ile Gly Gly Ser Val Ser Ala Gly 340 345
350Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His Ser
Leu Ser 355 360 365Leu Ala Gly Glu
Arg Thr Trp Ala Glu Thr Met Gly Leu Asn Thr Ala 370
375 380Asp Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val
Asn Thr Gly Thr385 390 395
400Ala Pro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val Leu Gly Lys
405 410 415Asn Gln Thr Leu Ala
Thr Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln 420
425 430Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn
Leu Ala Pro Ile 435 440 445Ala Leu
Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile Thr Met Asn 450
455 460Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys
Gln Leu Arg Leu Asp465 470 475
480Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly
485 490 495Arg Val Arg Val
Asp Thr Gly Ser Asn Trp Ser Glu Val Leu Pro Gln 500
505 510Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn
Gly Lys Asp Leu Asn 515 520 525Leu
Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser Asp Pro Leu Glu 530
535 540Thr Thr Lys Pro Asp Met Thr Leu Lys Glu
Ala Leu Lys Ile Ala Phe545 550 555
560Gly Phe Asn Glu Pro Asn Gly Asn Leu Gln Tyr Gln Gly Lys Asp
Ile 565 570 575Thr Glu Phe
Asp Phe Asn Phe Asp Gln Gln Thr Ser Gln Asn Ile Lys 580
585 590Asn Gln Leu Ala Glu Leu Asn Val Thr Asn
Ile Tyr Thr Val Leu Asp 595 600
605Lys Ile Lys Leu Asn Ala Lys Met Asn Ile Leu Ile Arg Asp Lys Arg 610
615 620Phe His Tyr Asp Arg Asn Asn Ile
Ala Val Gly Ala Asp Glu Ser Val625 630
635 640Val Lys Glu Ala His Arg Glu Val Ile Asn Ser Ser
Thr Glu Gly Leu 645 650
655Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly Tyr Ile
660 665 670Val Glu Ile Glu Asp Thr
Glu Gly Leu Lys Glu Val Ile Asn Asp Arg 675 680
685Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys
Thr Phe 690 695 700Ile Asp Phe Lys Lys
Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn705 710
715 720Pro Asn Tyr Lys Val Asn Val Tyr Ala Val
Thr Lys Glu Asn Thr Ile 725 730
735Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile Lys Lys
740 745 750Ile Leu Ile Phe Ser
Lys Lys Gly Tyr Glu Ile Gly 755 7606829PRTBacillus
anthracis 682Tyr Asn Val Leu Pro Thr Thr Ser Leu1
56839PRTBacillus anthracis 683Leu Ile Pro Leu Met Ala Leu Ser Thr1
56849PRTBacillus anthracis 684Tyr Val Asn Thr Gly Thr Ala Pro Ile1
56859PRTBacillus anthracis 685Tyr Ile Ser Asn Pro Asn Tyr
Lys Val1 56869PRTBacillus anthracis 686Leu Arg Gln Asp Gly
Lys Thr Phe Ile1 56879PRTBacillus anthracis 687Leu Ile Arg
Asp Lys Arg Phe His Tyr1 56889PRTBacillus anthracis 688Ile
Lys Leu Asn Ala Lys Met Asn Ile1 56899PRTBacillus anthracis
689Phe His Tyr Asp Arg Asn Asn Ile Ala1 56909PRTBacillus
anthracis 690Ile Ile Leu Ser Lys Asn Glu Asp Gln1
56919PRTBacillus anthracis 691Val Ile Ser Ser Asp Asn Leu Gln Leu1
56929PRTBacillus anthracis 692Val Ile Asn Ser Ser Thr Glu Gly Leu1
56939PRTBacillus anthracis 693Phe Lys Leu Tyr Trp Thr Asp
Ser Gln1 56949PRTBacillus anthracis 694Val Lys Asn Lys Arg
Thr Phe Leu Ser1 56959PRTBacillus anthracis 695Phe Ile Lys
Val Lys Lys Ser Asp Glu1 56969PRTBacillus anthracis 696Ile
Leu Ile Phe Ser Lys Lys Gly Tyr1 56979PRTBacillus anthracis
697Leu Leu Gly Tyr Tyr Phe Ser Asp Leu1 56989PRTBacillus
anthracis 698Ile Arg Lys Ile Leu Ser Gly Tyr Ile1
56999PRTBacillus anthracis 699Val Ala Ile Asp His Ser Leu Ser Leu1
57009PRTBacillus anthracis 700Ile Leu Ser Gly Tyr Ile Val Glu Ile1
57019PRTBacillus anthracis 701Ala Ile Trp Ser Gly Phe Ile
Lys Val1 57029PRTBacillus anthracis 702Phe Leu Ser Pro Trp
Ile Ser Asn Ile1 57039PRTBacillus anthracis 703Arg Leu Asn
Ala Asn Ile Arg Tyr Val1 57049PRTBacillus anthracis 704Asn
Ile Lys Asn Gln Leu Ala Glu Leu1 57059PRTBacillus anthracis
705Ser Leu Glu Val Glu Gly Tyr Thr Val1 57069PRTBacillus
anthracis 706Lys Leu Asn Ala Lys Met Asn Ile Leu1
57079PRTBacillus anthracis 707Tyr Ile Ser Asn Pro Asn Tyr Lys Val1
57089PRTBacillus anthracis 708Val Ala Ala Tyr Pro Ile Val His Val1
57099PRTBacillus anthracis 709Leu Val Leu Gly Lys Asn Gln
Thr Leu1 571012PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/anthrax protective antigen MHC
Class II epitope hybrid peptide 710Leu Arg Met Lys Asn Val Leu Pro
Thr Thr Ser Leu1 5 1071113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/anthrax
protective antigen MHC Class II epitope hybrid peptide 711Leu Arg
Met Lys Leu Ile Pro Leu Met Ala Leu Ser Thr1 5
1071212PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/anthrax protective antigen MHC Class II
epitope hybrid peptide 712Leu Arg Met Lys Val Asn Thr Gly Thr Ala Pro
Ile1 5 1071313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/anthrax
protective antigen MHC Class II epitope hybrid peptide 713Leu Arg
Met Lys Tyr Ile Ser Asn Pro Asn Tyr Lys Val1 5
1071413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/anthrax protective antigen MHC Class II
epitope hybrid peptide 714Leu Arg Met Lys Leu Arg Gln Asp Gly Lys Thr Phe
Ile1 5 1071513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/anthrax
protective antigen MHC Class II epitope hybrid peptide 715Leu Arg
Met Lys Leu Ile Arg Asp Lys Arg Phe His Tyr1 5
1071613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/anthrax protective antigen MHC Class II
epitope hybrid peptide 716Leu Arg Met Lys Ile Lys Leu Asn Ala Lys Met Asn
Ile1 5 1071713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/anthrax
protective antigen MHC Class II epitope hybrid peptide 717Leu Arg
Met Lys Phe His Tyr Asp Arg Asn Asn Ile Ala1 5
1071813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/anthrax protective antigen MHC Class II
epitope hybrid peptide 718Leu Arg Met Lys Ile Ile Leu Ser Lys Asn Glu Asp
Gln1 5 1071913PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/anthrax
protective antigen MHC Class II epitope hybrid peptide 719Leu Arg
Met Lys Val Ile Ser Ser Asp Asn Leu Gln Leu1 5
1072033PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/anthrax protective antigen MHC Class II
epitope/anthrax protective antigen ARD hybrid peptide 720Leu Arg Met
Lys Xaa Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu1 5
10 15Leu Lys Gln Lys Ser Ser Asn Ser Arg
Lys Lys Arg Ser Thr Ser Ala 20 25
30Gly72125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/anthrax protective antigen MHC Class II
epitope/anthrax protective antigen ARD hybrid peptide 721Leu Arg Met
Lys Pro Asp Ser Leu Glu Val Glu Gly Tyr Thr Val Asp1 5
10 15Val Lys Asn Lys Arg Thr Phe Leu Ser
20 2572234PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/anthrax protective antigen MHC
Class II epitope/anthrax protective antigen ARD hybrid peptide
722Leu Arg Met Lys Val Pro Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser1
5 10 15Leu Glu Val Glu Gly Tyr
Thr Val Asp Val Lys Asn Lys Arg Thr Phe 20 25
30Leu Ser72326PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/anthrax protective antigen MHC
Class II epitope/anthrax protective antigen ARD hybrid peptide
723Leu Arg Met Lys Xaa Ile Arg Lys Ile Leu Ser Gly Tyr Ile Val Glu1
5 10 15Ile Glu Asp Thr Glu Gly
Leu Lys Glu Val 20 2572425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/anthrax
protective antigen MHC Class II epitope/anthrax protective antigen
ARD hybrid peptide 724Leu Arg Met Lys Ile Asn Asp Arg Tyr Asp Met
Leu Asn Ile Ser Ser1 5 10
15Leu Arg Gln Asp Gly Lys Thr Phe Ile 20
2572513PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/anthrax protective antigen MHC Class II epitope/anthrax
protective antigen ARD hybrid peptide 725Leu Arg Met Lys Tyr Val Asn
Thr Gly Thr Ala Pro Ile1 5
1072613PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/anthrax protective antigen MHC Class II epitope/anthrax
protective antigen ARD hybrid peptide 726Leu Arg Met Lys Tyr Val Asn
Thr Gly Thr Ala Pro Ile1 5
1072714PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/anthrax protective antigen MHC Class II epitope/anthrax
protective antigen ARD hybrid peptide 727Leu Arg Met Lys Xaa Tyr Val
Asn Thr Gly Thr Ala Pro Ile1 5
1072814PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/anthrax protective antigen MHC Class II epitope/anthrax
protective antigen ARD hybrid peptide 728Leu Arg Met Lys Xaa Tyr Val
Asn Thr Gly Thr Ala Pro Ile1 5
1072914PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/anthrax protective antigen MHC Class II epitope/anthrax
protective antigen ARD hybrid peptide 729Leu Arg Met Lys Xaa Tyr Val
Asn Thr Gly Thr Ala Pro Ile1 5
1073014PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/anthrax protective antigen MHC Class II epitope/anthrax
protective antigen ARD hybrid peptide 730Leu Arg Met Lys Xaa Tyr Val
Asn Thr Gly Thr Ala Pro Ile1 5
1073113PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/anthrax protective antigen MHC Class II epitope/anthrax
protective antigen ARD hybrid peptide 731Leu Arg Met Lys Tyr Val Asn
Thr Gly Thr Ala Pro Ile1 5
1073221PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/anthrax protective antigen MHC Class II epitope/anthrax
protective antigen ARD hybrid peptide 732Leu Arg Met Lys Asn Gly Ile
Lys Lys Ile Leu Ile Phe Ser Lys Lys1 5 10
15Gly Tyr Glu Ile Gly 20733317PRTVariola
virus 733Met Lys Thr Ile Ser Val Val Thr Leu Leu Cys Val Leu Pro Ala Val1
5 10 15Val Tyr Ser Thr
Cys Thr Val Pro Thr Met Asn Asn Ala Lys Leu Thr 20
25 30Ser Thr Glu Thr Ser Phe Asn Asp Lys Gln Lys
Val Thr Phe Thr Cys 35 40 45Asp
Ser Gly Tyr Tyr Ser Leu Asp Pro Asn Ala Val Cys Glu Thr Asp 50
55 60Lys Trp Lys Tyr Glu Asn Pro Cys Lys Lys
Met Cys Thr Val Ser Asp65 70 75
80Tyr Val Ser Glu Leu Tyr Asn Lys Pro Leu Tyr Glu Val Asn Ala
Ile 85 90 95Ile Thr Leu
Ile Cys Lys Asp Glu Thr Lys Tyr Phe Arg Cys Glu Glu 100
105 110Lys Asn Gly Asn Thr Ser Trp Asn Asp Thr
Val Thr Cys Pro Asn Ala 115 120
125Glu Cys Gln Ser Leu Gln Leu Asp His Gly Ser Cys Gln Pro Val Lys 130
135 140Glu Lys Tyr Ser Phe Gly Glu His
Ile Thr Ile Asn Cys Asp Val Gly145 150
155 160Tyr Glu Val Ile Gly Ala Ser Tyr Ile Thr Cys Thr
Ala Asn Ser Trp 165 170
175Asn Val Ile Pro Ser Cys Gln Gln Lys Cys Asp Ile Pro Ser Leu Ser
180 185 190Asn Gly Leu Ile Ser Gly
Ser Thr Phe Ser Ile Gly Gly Val Ile His 195 200
205Leu Ser Cys Lys Ser Gly Phe Ile Leu Thr Gly Ser Pro Ser
Ser Thr 210 215 220Cys Ile Asp Gly Lys
Trp Asn Pro Val Leu Pro Ile Cys Ile Arg Ser225 230
235 240Asn Glu Glu Phe Asp Pro Val Glu Asp Gly
Pro Asp Asp Glu Thr Asp 245 250
255Leu Ser Lys Leu Ser Lys Asp Val Val Gln Tyr Glu Gln Glu Ile Glu
260 265 270Ser Leu Glu Ala Thr
Tyr His Ile Ile Ile Val Ala Leu Thr Ile Met 275
280 285Gly Val Ile Phe Leu Ile Ser Val Ile Val Leu Val
Cys Ser Cys Asn 290 295 300Lys Asn Asn
Asp Gln Tyr Lys Phe His Lys Leu Leu Leu305 310
3157349PRTVariola virus 734Val Ile Phe Leu Ile Ser Val Ile Val1
57359PRTVariola virus 735Ile Phe Leu Ile Ser Val Ile Val Leu1
57369PRTVariola virus 736Phe Leu Ile Ser Val Ile Val Leu Val1
57379PRTVariola virus 737Tyr Tyr Ser Leu Asp Pro Asn Ala
Val1 57389PRTVariola virus 738Trp Asn Pro Val Leu Pro Ile
Cys Ile1 57399PRTVariola virus 739Ile His Leu Ser Cys Lys
Ser Gly Phe1 57409PRTVariola virus 740Ile Val Ala Leu Thr
Ile Met Gly Val1 57419PRTVariola virus 741Ile Ile Ile Val
Ala Leu Thr Ile Met1 57429PRTVariola virus 742Phe Ile Leu
Thr Gly Ser Pro Ser Ser1 57439PRTVariola virus 743Trp Asn
Val Ile Pro Ser Cys Gln Gln1 57449PRTVariola virus 744Tyr
Ser Leu Asp Pro Asn Ala Val Cys1 57459PRTVariola virus
745Tyr His Ile Ile Ile Val Ala Leu Thr1 57469PRTVariola
virus 746Leu Thr Ile Met Gly Val Ile Phe Leu1
57479PRTVariola virus 747Val Thr Leu Leu Cys Val Leu Pro Ala1
57489PRTVariola virus 748Leu Tyr Asn Lys Pro Leu Tyr Glu Val1
57499PRTVariola virus 749Val Ile Phe Leu Ile Ser Val Ile Val1
575016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/variola B5R epitope hybrid peptide 750Leu Arg Met
Lys Xaa Val Ile Phe Leu Ile Ser Val Ile Val Leu Val1 5
10 1575114PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/variola B5R epitope hybrid
peptide 751Leu Arg Met Lys Xaa Tyr Tyr Ser Leu Asp Pro Asn Ala Val1
5 1075214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/variola B5R epitope hybrid
peptide 752Leu Arg Met Lys Xaa Trp Asn Pro Val Leu Pro Ile Cys Ile1
5 1075314PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/variola B5R epitope hybrid
peptide 753Leu Arg Met Lys Xaa Ile His Leu Ser Cys Lys Ser Gly Phe1
5 1075416PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/variola B5R epitope hybrid
peptide 754Leu Arg Met Lys Xaa Ile Ile Ile Val Ala Leu Thr Ile Met Gly
Val1 5 10
1575514PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/variola B5R epitope hybrid peptide 755Leu Arg Met Lys Xaa Phe
Ile Leu Thr Gly Ser Pro Ser Ser1 5
1075614PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/variola B5R epitope hybrid peptide 756Leu Arg Met Lys Xaa Trp
Asn Val Ile Pro Ser Cys Gln Gln1 5
1075714PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/variola B5R epitope hybrid peptide 757Leu Arg Met Lys Xaa Tyr
His Ile Ile Ile Val Ala Leu Thr1 5
1075814PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/variola B5R epitope hybrid peptide 758Leu Arg Met Lys Xaa Tyr
His Ile Ile Ile Val Ala Leu Thr1 5
1075914PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/variola B5R epitope hybrid peptide 759Leu Arg Met Lys Xaa Leu
Thr Ile Met Gly Val Ile Phe Leu1 5
1076014PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/variola B5R epitope hybrid peptide 760Leu Arg Met Lys Xaa Val
Thr Leu Leu Cys Val Leu Pro Ala1 5
1076114PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/variola B5R epitope hybrid peptide 761Leu Arg Met Lys Xaa Leu
Tyr Asn Lys Pro Leu Tyr Glu Val1 5
1076214PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/variola B5R epitope hybrid peptide 762Leu Arg Met Lys Xaa Val
Ile Phe Leu Ile Ser Val Ile Val1 5
107639PRTVariola virus 763Phe Leu Ile Ser Val Ile Val Leu Val1
57649PRTVariola virus 764Thr Leu Leu Cys Val Leu Pro Ala Val1
57659PRTVariola virus 765Lys Met Cys Thr Val Ser Asp Tyr Val1
57669PRTVariola virus 766Thr Ile Met Gly Val Ile Phe Leu Ile1
57679PRTVariola virus 767Leu Leu Cys Val Leu Pro Ala Val Val1
57689PRTVariola virus 768Val Leu Pro Ala Val Val Tyr Ser Thr1
57699PRTVariola virus 769Val Ile Phe Leu Ile Ser Val Ile
Val1 57709PRTVariola virus 770Ile Val Ala Leu Thr Ile Met
Gly Val1 57719PRTVariola virus 771Thr Val Ser Asp Tyr Val
Ser Glu Leu1 57729PRTVariola virus 772Leu Ile Ser Gly Ser
Thr Phe Ser Ile1 577316PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/MHC Class II-presented/MHC
Class I-presented B5R hybrid peptide 773Leu Arg Met Lys Xaa Val Ile
Phe Leu Ile Ser Val Ile Val Leu Val1 5 10
1577420PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/MHC Class II-presented/MHC Class
I-presented B5R hybrid peptide 774Leu Arg Met Lys Xaa Thr Ile Met
Gly Val Ile Phe Leu Ile Ser Val1 5 10
15Ile Val Leu Val 2077522PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/MHC
Class II-presented/MHC Class I-presented B5R hybrid peptide 775Leu
Arg Met Lys Xaa Tyr His Ile Ile Ile Val Ala Leu Thr Ile Met1
5 10 15Gly Val Ile Phe Leu Ile
2077616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MHC Class II-presented/MHC Class I-presented
B5R hybrid peptide 776Leu Arg Met Lys Xaa Val Thr Leu Leu Cys Val Leu Pro
Ala Val Val1 5 10
15777251PRTEbola virus 777Met Ala Lys Ala Thr Gly Arg Tyr Asn Leu Ile Ser
Pro Lys Lys Asp1 5 10
15Leu Glu Lys Gly Val Val Leu Ser Asp Leu Cys Asn Phe Leu Val Ser
20 25 30Gln Thr Ile Gln Gly Trp Lys
Val Tyr Trp Ala Gly Ile Glu Phe Asp 35 40
45Val Thr His Lys Gly Met Ala Leu Leu His Arg Leu Lys Thr Asn
Asp 50 55 60Phe Ala Pro Ala Trp Ser
Met Thr Arg Asn Leu Phe Pro His Leu Phe65 70
75 80Gln Asn Pro Asn Ser Thr Ile Glu Ser Pro Leu
Trp Ala Leu Arg Val 85 90
95Ile Leu Ala Ala Gly Ile Gln Asp Gln Leu Ile Asp Gln Ser Leu Ile
100 105 110Glu Pro Leu Ala Gly Ala
Leu Gly Leu Ile Ser Asp Trp Leu Leu Thr 115 120
125Thr Asn Thr Asn His Phe Asn Met Arg Thr Gln Arg Val Lys
Glu Gln 130 135 140Leu Ser Leu Lys Met
Leu Ser Leu Ile Arg Ser Asn Ile Leu Lys Phe145 150
155 160Ile Asn Lys Leu Asp Ala Leu His Val Val
Asn Tyr Asn Gly Leu Leu 165 170
175Ser Ser Ile Glu Ile Gly Thr Gln Asn His Thr Ile Ile Ile Thr Arg
180 185 190Thr Asn Met Gly Phe
Leu Val Glu Leu Gln Glu Pro Asp Lys Ser Ala 195
200 205Met Asn Arg Lys Lys Pro Gly Pro Ala Lys Phe Ser
Leu Leu His Glu 210 215 220Ser Thr Leu
Lys Ala Phe Thr Gln Gly Ser Ser Thr Arg Met Gln Ser225
230 235 240Leu Ile Leu Glu Phe Asn Ser
Ser Leu Ala Ile 245 2507789PRTEbola virus
778Leu Arg Val Ile Leu Ala Ala Gly Ile1 57799PRTEbola virus
779Val Val Leu Ser Asp Leu Cys Asn Phe1 57809PRTEbola virus
780Leu Ile Arg Ser Asn Ile Leu Lys Phe1 57819PRTEbola virus
781Leu Lys Met Leu Ser Leu Ile Arg Ser1 57829PRTEbola virus
782Leu Lys Phe Ile Asn Lys Leu Asp Ala1 57839PRTEbola virus
783Met Thr Arg Gln Arg Val Lys Glu Gln1 57849PRTEbola virus
784Val Asn Tyr Asn Gly Leu Leu Ser Ser1 57859PRTEbola virus
785Leu Leu His Glu Ser Thr Leu Lys Ala1 57869PRTEbola virus
786Trp Leu Leu Thr Thr Asn Thr Asn His1 57879PRTEbola virus
787Ile Ile Ile Thr Arg Thr Asn Met Gly1 57889PRTEbola virus
788Phe Leu Val Ser Gln Thr Ile Gln Gly1 57899PRTEbola virus
789Ile Gln Gly Trp Lys Val Tyr Trp Ala1 57905PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key motif
790Leu Ile Val Phe Met1 579113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ebola
virus VP24 MHC Class II epitope hybrid peptide 791Leu Arg Met Lys
Val Val Leu Ser Asp Leu Cys Asn Phe1 5
1079213PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ebola virus VP24 MHC Class II epitope hybrid peptide
792Leu Arg Met Lys Phe Leu Val Ser Gln Thr Ile Gln Gly1 5
1079313PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ebola virus VP24 MHC Class II epitope
hybrid peptide 793Leu Arg Met Lys Ile Gln Gly Trp Lys Val Tyr Trp Ala1
5 1079419PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/Ebola virus VP24 MHC Class
II epitope hybrid peptide 794Leu Arg Met Lys Phe Leu Val Ser Gln Thr
Ile Gln Gly Trp Lys Val1 5 10
15Tyr Trp Ala79513PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ebola virus VP24 MHC Class II epitope
hybrid peptide 795Leu Arg Met Lys Leu Lys Met Leu Ser Leu Ile Arg Ser1
5 1079613PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Ii-key/Ebola virus VP24 MHC Class
II epitope hybrid peptide 796Leu Arg Met Lys Leu Ile Arg Ser Asn Ile
Leu Lys Phe1 5 1079713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ebola
virus VP24 MHC Class II epitope hybrid peptide 797Leu Arg Met Lys
Leu Lys Phe Ile Asn Lys Leu Asp Ala1 5
1079818PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/Ebola virus VP24 MHC Class II epitope hybrid peptide
798Leu Arg Met Lys Leu Lys Met Leu Ser Leu Ile Arg Ser Asn Ile Leu1
5 10 15Lys
Phe79913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Ebola virus VP24 MHC Class II epitope hybrid
peptide 799Leu Arg Met Lys Val Asn Tyr Asn Gly Leu Leu Ser Ser1
5 1080013PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Ebola virus VP24 MHC Class II
epitope hybrid peptide 800Leu Arg Met Lys Leu Leu His Glu Ser Thr
Leu Lys Ala1 5 108014PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ebola
virus VP24 MHC Class II epitope hybrid peptide 801Leu Arg Met
Lys18029PRTEbola virus 802Val Leu Ser Asp Leu Cys Asn Phe Leu1
58039PRTEbola virus 803Leu Ile Leu Glu Phe Asn Ser Ser Leu1
58049PRTEbola virus 804Asn Ile Leu Lys Phe Ile Asn Lys Leu1
58059PRTEbola virus 805Ala Leu Gly Leu Ile Ser Asp Trp Leu1
58069PRTEbola virus 806Ser Gln Thr Ile Gln Gly Trp Lys Val1
58079PRTEbola virus 807Gln Leu Ser Leu Lys Met Leu Ser Leu1
58089PRTEbola virus 808Ser Leu Ile Glu Pro Leu Ala Gly Ala1
58099PRTEbola virus 809Leu Leu His Glu Ser Thr Leu Lys Ala1
58109PRTEbola virus 810Asn Leu Ile Ser Pro Lys Lys Asp Leu1
58119PRTEbola virus 811Met Leu Ser Leu Ile Arg Ser Asn Ile1
58129PRTEbola virus 812Leu Ile Ser Asp Trp Leu Leu Thr Thr1
58139PRTEbola virus 813Gly Leu Ile Ser Asp Trp Leu Leu Thr1
581414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Ebola VP24 MHC Class II-predicted
epitope/Ebola VP24 MHC Class I-predicted epitope hybrid peptide
814Leu Arg Met Lys Val Val Leu Ser Asp Leu Cys Asn Phe Leu1
5 1081520PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/Ebola VP24 MHC Class
II-predicted epitope/Ebola VP24 MHC Class I-predicted epitope
hybrid peptide 815Leu Arg Met Lys Val Leu Ser Asp Leu Cys Asn Phe Leu Val
Ser Gln1 5 10 15Thr Ile
Gln Gly 2081618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ebola VP24 MHC Class II-predicted
epitope/Ebola VP24 MHC Class I-predicted epitope hybrid peptide
816Leu Arg Met Lys Gly Leu Ile Ser Asp Trp Leu Leu Thr Thr Asn Thr1
5 10 15Asn
His81715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/Ebola VP24 MHC Class II-predicted
epitope/Ebola VP24 MHC Class I-predicted epitope hybrid peptide
817Leu Arg Met Lys Gln Leu Ser Leu Lys Met Leu Ser Ile Arg Ser1
5 10 1581818PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ebola
VP24 MHC Class II-predicted epitope/Ebola VP24 MHC Class I-predicted
epitope hybrid peptide 818Leu Arg Met Lys Gln Leu Ser Leu Lys Met
Leu Ser Leu Ile Arg Ser1 5 10
15Asn Ile81915PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ebola VP24 MHC Class II-predicted
epitope/Ebola VP24 MHC Class I-predicted epitope hybrid peptide
819Leu Arg Met Lys Asn Ile Leu Lys Phe Ile Asn Lys Leu Asp Ala1
5 10 1582019PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/Ebola
VP24 MHC Class II-predicted epitope/Ebola VP24 MHC Class I-predicted
epitope hybrid peptide 820Leu Arg Met Lys Leu Ile Arg Ser Asn Ile
Leu Lys Phe Ile Asn Lys1 5 10
15Leu Asp Ala82113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/Ebola VP24 MHC Class II-predicted
epitope/Ebola VP24 MHC Class I-predicted epitope hybrid peptide
821Leu Arg Met Lys Leu Leu His Glu Ser Thr Leu Lys Ala1 5
1082212PRTHomo sapiens 822Pro Val Val His Phe Phe Lys Asn
Ile Val Thr Pro1 5 1082316PRTUnknown
OrganismDescription of Unknown Organism PL139-154 peptide 823His Cys
Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe Val Gly Ile1 5
10 15824304PRTHomo sapiens 824Met Gly
Asn His Ala Gly Lys Arg Glu Leu Asn Ala Glu Lys Ala Ser1 5
10 15Thr Asn Ser Glu Thr Asn Arg Gly
Glu Ser Glu Lys Lys Arg Asn Leu 20 25
30Gly Glu Leu Ser Arg Thr Thr Ser Glu Asp Asn Glu Val Phe Gly
Glu 35 40 45Ala Asp Ala Asn Gln
Asn Asn Gly Thr Ser Ser Gln Asp Thr Ala Val 50 55
60Thr Asp Ser Lys Arg Thr Ala Asp Pro Lys Asn Ala Trp Gln
Asp Ala65 70 75 80His
Pro Ala Asp Pro Gly Ser Arg Pro His Leu Ile Arg Leu Phe Ser
85 90 95Arg Asp Ala Pro Gly Arg Glu
Asp Asn Thr Phe Lys Asp Arg Pro Ser 100 105
110Glu Ser Asp Glu Leu Gln Thr Ile Gln Glu Asp Ser Ala Ala
Thr Ser 115 120 125Glu Ser Leu Asp
Val Met Ala Ser Gln Lys Arg Pro Ser Gln Arg His 130
135 140Gly Ser Lys Tyr Leu Ala Thr Ala Ser Thr Met Asp
His Ala Arg His145 150 155
160Gly Phe Leu Pro Arg His Arg Asp Thr Gly Ile Leu Asp Ser Ile Gly
165 170 175Arg Phe Phe Gly Gly
Asp Arg Gly Ala Pro Lys Arg Gly Ser Gly Lys 180
185 190Asp Ser His His Pro Ala Arg Thr Ala His Tyr Gly
Ser Leu Pro Gln 195 200 205Lys Ser
His Gly Arg Thr Gln Asp Glu Asn Pro Val Val His Phe Phe 210
215 220Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro
Ser Gln Gly Lys Gly225 230 235
240Arg Gly Leu Ser Leu Ser Arg Phe Ser Trp Gly Ala Glu Gly Gln Arg
245 250 255Pro Gly Phe Gly
Tyr Gly Gly Arg Ala Ser Asp Tyr Lys Ser Ala His 260
265 270Lys Gly Phe Lys Gly Val Asp Ala Gln Gly Thr
Leu Ser Lys Ile Phe 275 280 285Lys
Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Pro Met Ala Arg Arg 290
295 3008259PRTHomo sapiens 825Leu Ser Lys Ile
Phe Lys Leu Gly Gly1 58269PRTHomo sapiens 826Arg Pro His
Leu Ile Arg Leu Phe Ser1 58279PRTHomo sapiens 827His Ala
Gly Lys Arg Glu Leu Asn Ala1 58289PRTHomo sapiens 828His
Lys Gly Phe Lys Gly Val Asp Ala1 58299PRTHomo sapiens
829Leu Gln Thr Ile Gln Glu Asp Ser Ala1 58309PRTHomo
sapiens 830Arg Asp Thr Gly Ile Leu Asp Ser Ile1
58319PRTHomo sapiens 831Asp Ser Lys Arg Thr Ala Asp Pro Lys1
58329PRTHomo sapiens 832Val His Phe Phe Lys Asn Ile Val Thr1
58339PRTHomo sapiens 833Ala Ser Thr Met Asp His Ala Arg His1
58349PRTHomo sapiens 834Lys Arg Asn Leu Gly Glu Leu Ser Arg1
58359PRTHomo sapiens 835Gly Arg Phe Phe Gly Gly Asp Arg Gly1
583644PRTHomo sapiens 836Met Gly Asn His Ala Gly Lys Arg Glu Leu Asn
Ala Glu Lys Ala Ser1 5 10
15Thr Asn Ser Glu Thr Asn Arg Gly Glu Ser Glu Lys Lys Arg Asn Leu
20 25 30Gly Glu Leu Ser Arg Thr Thr
Ser Glu Asp Asn Glu 35 4083736PRTHomo sapiens
837Ala Trp Gln Asp Ala His Pro Ala Asp Pro Gly Ser Arg Pro His Leu1
5 10 15Ile Arg Leu Phe Ser Arg
Asp Ala Pro Gly Arg Glu Asp Asn Thr Phe 20 25
30Lys Asp Arg Pro 3583815PRTHomo sapiens 838Leu
Asp Val Met Ala Ser Gln Lys Arg Pro Ser Gln Arg His Gly1 5
10 1583916PRTHomo sapiens 839Pro Ser
Gln Arg His Gly Ser Lys Tyr Leu Ala Thr Ala Ser Thr Met1 5
10 1584020PRTHomo sapiens 840Ala His
Pro Ala Asp Pro Gly Ser Arg Pro His Leu Ile Arg Leu Phe1 5
10 15Ser Arg Asp Ala
2084114PRTHomo sapiens 841Leu Ala Thr Ala Ser Thr Met Asp His Ala Arg His
Gly Phe1 5 108429PRTHomo sapiens 842Phe
Ile Arg Leu Phe Ser Arg Asp Ala1 58439PRTHomo sapiens
843Ile Arg Leu Phe Ser Arg Asp Ala Pro1 58449PRTHomo
sapiens 844Ile Gln Trp Asp Ser Ala Ala Thr Ala1
58459PRTHomo sapiens 845Val Met Ala Ser Gln Lys Arg Pro Ser1
58469PRTHomo sapiens 846Leu Ala Thr Ala Ser Thr Met Asp His1
584713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/human MBP antigentic epitope hybrid peptide
847Leu Arg Met Lys Leu Ser Lys Ile Phe Lys Leu Gly Gly1 5
1084813PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/human MBP antigentic epitope hybrid
peptide 848Leu Arg Met Lys Arg Pro His Leu Ile Arg Leu Phe Ser1
5 1084913PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/human MBP antigentic epitope
hybrid peptide 849Leu Arg Met Lys His Ala Gly Lys Arg Glu Leu Asn
Ala1 5 1085013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/human
MBP antigentic epitope hybrid peptide 850Leu Arg Met Lys His Lys Gly
Phe Lys Gly Val Asp Ala1 5
1085113PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/human MBP antigentic epitope hybrid peptide 851Leu Arg
Met Lys His Lys Gly Phe Lys Gly Val Asp Ala1 5
1085213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/human MBP antigentic epitope hybrid peptide
852Leu Arg Met Lys Phe Ile Arg Leu Phe Ser Arg Asp Ala1 5
1085313PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/human MBP antigentic epitope hybrid
peptide 853Leu Arg Met Lys Ile Arg Leu Phe Ser Arg Asp Ala Pro1
5 1085413PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/human MBP antigentic epitope
hybrid peptide 854Leu Arg Met Lys Val Met Ala Ser Gln Lys Arg Pro
Ser1 5 1085513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Ii-key/human
MBP antigentic epitope hybrid peptide 855Leu Arg Met Lys Leu Ala Thr
Ala Ser Thr Met Asp His1 5
1085616PRTMurine sp. 856His Cys Leu Gly Lys Trp Leu Gly His Pro Asp Lys
Phe Val Gly Ile1 5 10
1585714PRTHomo sapiens 857Phe Asn Thr Trp Thr Thr Cys Asp Ser Ile Ala Phe
Pro Ser1 5 10858277PRTHomo sapiens 858Met
Gly Leu Leu Glu Cys Cys Ala Arg Cys Leu Val Gly Ala Pro Phe1
5 10 15Ala Ser Leu Val Ala Thr Gly
Leu Cys Phe Phe Gly Val Ala Leu Phe 20 25
30Cys Gly Cys Gly His Glu Ala Leu Thr Gly Thr Glu Lys Leu
Ile Glu 35 40 45Thr Tyr Phe Ser
Lys Asn Tyr Gln Asp Tyr Glu Tyr Leu Ile Asn Val 50 55
60Ile His Ala Phe Gln Tyr Val Ile Tyr Gly Thr Ala Ser
Phe Phe Phe65 70 75
80Leu Tyr Gly Ala Leu Leu Leu Ala Glu Gly Phe Tyr Thr Thr Gly Ala
85 90 95Val Arg Gln Ile Phe Gly
Asp Tyr Lys Thr Thr Ile Cys Gly Lys Gly 100
105 110Leu Ser Ala Thr Val Thr Gly Gly Gln Lys Gly Arg
Gly Ser Arg Gly 115 120 125Gln His
Gln Ala His Ser Leu Glu Arg Val Cys His Cys Leu Gly Lys 130
135 140Trp Leu Gly His Pro Asp Lys Phe Val Gly Ile
Thr Tyr Ala Leu Thr145 150 155
160Val Val Trp Leu Leu Val Phe Ala Cys Ser Ala Val Pro Val Tyr Ile
165 170 175Tyr Phe Asn Thr
Trp Thr Thr Cys Asp Ser Ile Ala Phe Pro Ser Lys 180
185 190Thr Ser Ala Ser Ile Gly Ser Leu Cys Ala Asp
Ala Arg Met Tyr Gly 195 200 205Val
Leu Pro Trp Ile Ala Phe Pro Gly Lys Val Cys Gly Ser Asn Leu 210
215 220Leu Ser Ile Cys Lys Thr Ala Glu Phe Gln
Met Thr Phe His Leu Phe225 230 235
240Ile Ala Ala Phe Val Gly Ala Ala Ala Thr Leu Val Ser Leu Leu
Thr 245 250 255Phe Met Ile
Ala Ala Thr Tyr Asn Phe Ala Val Leu Lys Leu Met Gly 260
265 270Arg Gly Thr Lys Phe 2758599PRTHomo
sapiens 859Phe Phe Phe Leu Tyr Gly Ala Leu Leu1
58609PRTHomo sapiens 860Phe Val Gly Ala Ala Ala Thr Leu Val1
58619PRTHomo sapiens 861Phe His Leu Phe Ile Ala Ala Phe Val1
58629PRTHomo sapiens 862Leu Val Ser Leu Leu Thr Phe Met Ile1
58639PRTHomo sapiens 863Trp Leu Leu Val Phe Ala Cys Ser Ala1
58649PRTHomo sapiens 864Ile Phe Gly Asp Tyr Lys Thr Thr Ile1
58659PRTHomo sapiens 865Leu Cys Ala Asp Ala Arg Met Tyr Gly1
58669PRTHomo sapiens 866Val Ile Tyr Gly Thr Ala Ser Phe Phe1
58679PRTHomo sapiens 867Tyr Glu Tyr Leu Ile Asn Val Ile His1
58689PRTHomo sapiens 868Val Gly Ile Thr Tyr Ala Leu Thr Val1
586916PRTHomo sapiens 869His Cys Leu Gly Lys Trp Leu Gly His Pro
Asp Lys Phe Val Gly Ile1 5 10
1587018PRTHomo sapiens 870Phe Val Gly Ile Thr Tyr Ala Leu Thr Val
Val Trp Leu Leu Val Phe1 5 10
15Ala Cys8715PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key motif 871Leu Gly Lys Trp Leu1
587213PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/PLP epitope hybrid peptide 872Leu Arg Met Lys Phe Phe Phe Leu
Tyr Gly Ala Leu Leu1 5
1087313PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/PLP epitope hybrid peptide 873Leu Arg Met Lys Phe Val Gly Ala
Ala Ala Thr Leu Val1 5
1087413PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/PLP epitope hybrid peptide 874Leu Arg Met Lys Phe His Leu Phe
Ile Ala Ala Phe Val1 5
1087513PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/PLP epitope hybrid peptide 875Leu Arg Met Lys Leu Val Ser Leu
Leu Thr Phe Met Ile1 5
1087622PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/PLP epitope hybrid peptide 876Leu Arg Met Lys Phe Val Gly Ile
Thr Tyr Ala Leu Thr Val Val Trp1 5 10
15Leu Leu Val Phe Ala Cys 20877247PRTHomo sapiens
877Met Ala Ser Leu Ser Arg Pro Ser Leu Pro Ser Cys Leu Cys Ser Phe1
5 10 15Leu Leu Leu Leu Leu Leu
Gln Val Ser Ser Ser Tyr Ala Gly Gln Phe 20 25
30Arg Val Ile Gly Pro Arg His Pro Ile Arg Ala Leu Val
Gly Asp Glu 35 40 45Val Glu Leu
Pro Cys Arg Ile Ser Pro Gly Lys Asn Ala Thr Gly Met 50
55 60Glu Val Gly Trp Tyr Arg Pro Pro Phe Ser Arg Val
Val His Leu Tyr65 70 75
80Arg Asn Gly Lys Asp Gln Asp Gly Asp Gln Ala Pro Glu Tyr Arg Gly
85 90 95Arg Thr Glu Leu Leu Lys
Asp Ala Ile Gly Glu Gly Lys Val Thr Leu 100
105 110Arg Ile Arg Asn Val Arg Phe Ser Asp Glu Gly Gly
Phe Thr Cys Phe 115 120 125Phe Arg
Asp His Ser Tyr Gln Glu Glu Ala Ala Met Glu Leu Lys Val 130
135 140Glu Asp Pro Phe Tyr Trp Val Ser Pro Gly Val
Leu Val Leu Leu Ala145 150 155
160Val Leu Pro Val Leu Leu Leu Gln Ile Thr Val Gly Leu Val Phe Leu
165 170 175Cys Leu Gln Tyr
Arg Leu Arg Gly Lys Leu Arg Ala Glu Ile Glu Asn 180
185 190Leu His Arg Thr Phe Asp Pro His Phe Leu Arg
Val Pro Cys Trp Lys 195 200 205Ile
Thr Leu Phe Val Ile Val Pro Val Leu Gly Pro Leu Val Ala Leu 210
215 220Ile Ile Cys Tyr Asn Trp Leu His Arg Arg
Leu Ala Gly Gln Phe Leu225 230 235
240Glu Glu Leu Arg Asn Pro Phe 2458789PRTHomo
sapiens 878Leu Val Leu Leu Ala Val Leu Pro Val1
58799PRTHomo sapiens 879Phe Leu Arg Val Pro Cys Trp Lys Ile1
58809PRTHomo sapiens 880Phe Arg Val Ile Gly Pro Arg His Pro1
58819PRTHomo sapiens 881Leu Gly Pro Leu Val Ala Leu Ile Ile1
58829PRTHomo sapiens 882Phe Val Ile Val Pro Val Leu Gly Pro1
58839PRTHomo sapiens 883Phe Leu Leu Leu Leu Leu Leu Gln Val1
58849PRTHomo sapiens 884Leu Val Gly Asp Glu Val Glu Leu Pro1
58859PRTHomo sapiens 885Leu Leu Lys Asp Ala Ile Gly Glu Gly1
58869PRTHomo sapiens 886Leu Arg Ile Arg Asn Val Arg Phe Ser1
58879PRTHomo sapiens 887Leu Leu Leu Gln Ile Thr Val Gly Leu1
58889PRTHomo sapiens 888Trp Val Ser Pro Gly Val Leu Val Leu1
58899PRTHomo sapiens 889Tyr Arg Leu Arg Gly Lys Leu Arg Ala1
58909PRTHomo sapiens 890Trp Leu His Arg Arg Leu Ala Gly Gln1
589119PRTHomo sapiens 891Leu Leu Gln Val Ser Ser Ser Tyr Ala
Gly Gln Phe Arg Val Ile Gly1 5 10
15Pro Arg His89213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/MOG epitope hybrid peptide
892Leu Arg Met Lys Leu Val Leu Leu Ala Val Leu Pro Val1 5
1089313PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/MOG epitope hybrid peptide 893Leu Arg Met
Lys Phe Leu Arg Val Pro Cys Trp Lys Ile1 5
1089413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Ii-key/MOG epitope hybrid peptide 894Leu Arg Met Lys Phe
Arg Val Ile Gly Pro Arg His Pro1 5
1089513PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/MOG epitope hybrid peptide 895Leu Arg Met Lys Phe Val Ile Val
Pro Val Leu Gly Pro1 5
1089617PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/MOG epitope hybrid peptide 896Leu Arg Met Lys Phe Leu Leu Leu
Leu Leu Leu Gln Val Ser Ser Ser1 5 10
15Tyr89716PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Ii-key/MOG epitope hybrid peptide 897Leu Arg Met
Lys Phe Arg Val Ile Gly Pro Arg His Pro Ile Arg Ala1 5
10 1589816PRTHuman immunodeficiency virus
type 1 898Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ser Gln
Ile1 5 10
1589920PRTHuman immunodeficiency virus type 1 899Tyr Pro Gly Ile Lys Val
Arg Gln Leu Cys Lys Leu Leu Arg Gly Thr1 5
10 15Lys Ala Leu Thr 2090015PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/antigenic epitope hybrid peptide 900Leu Arg Met Lys Xaa Ile Ala
Tyr Leu Lys Gln Ala Thr Ala Lys1 5 10
1590110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic antigenic epitope peptide 901Ile Ala Tyr Leu Lys
Gln Ala Thr Ala Lys1 5
1090216PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/antigenic epitope hybrid peptide 902Leu Arg Met Lys Xaa Xaa
Ala Tyr Arg Ala Ile Arg His Ile Pro Arg1 5
10 1590315PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Ii-key/antigenic epitope hybrid
peptide 903Leu Arg Met Lys Xaa Ala Tyr Arg Ala Ile Arg His Ile Pro Arg1
5 10 1590414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
Ii-key/antigenic epitope hybrid peptide 904Leu Arg Met Lys Xaa Tyr Arg
Ala Ile Arg His Ile Pro Arg1 5
1090514PRTArtificial SequenceDescription of Artificial Sequence Synthetic
Ii-key/antigenic epitope hybrid peptide 905Leu Arg Met Lys Ala Tyr
Arg Ala Ile Arg His Ile Pro Arg1 5 10
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