Patent application title: ANTI-HYDROXYLASE ANTIBODIES AND USES THEREOF
Inventors:
K. Dane Wittrup (Chestnut Hill, MA, US)
Jack R. Wands (Providence, RI, US)
Yik Andy Yeung (Pittsburg, CA, US)
IPC8 Class: AC07K1600FI
USPC Class:
5303891
Class name: Globulins immunoglobulin, antibody, or fragment thereof, other than immunoglobulin antibody, or fragment thereof that is conjugated or adsorbed polyclonal antibody or immunogloblin of identified binding specificity
Publication date: 2010-01-14
Patent application number: 20100010203
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Patent application title: ANTI-HYDROXYLASE ANTIBODIES AND USES THEREOF
Inventors:
K. Dane Wittrup
Jack R. Wands
Yik Andy Yeung
Agents:
FISH & RICHARDSON PC
Assignees:
Origin: MINNEAPOLIS, MN US
IPC8 Class: AC07K1600FI
USPC Class:
5303891
Patent application number: 20100010203
Abstract:
Antibodies, or antigen-binding portions thereof, to aspartyl (asparaginyl)
β-hydroxylase are provided. The anti-aspartyl (asparaginyl)
β-hydroxylase antibodies, or antigen-binding portions thereof, can
modulate activity of aspartyl (asparaginyl) β-hydroxylase.Claims:
1. A composition comprising an isolated human antibody or a fragment or
other variant thereof, wherein the antibody, the fragment, or the other
variant specifically binds to an aspartyl (asparaginyl)
β-hydroxylase (AAH).Description:
[0001]This application is a continuation of U.S. application Ser. No.
12/055,108 filed Mar. 25, 2008, which is a continuation of U.S.
application Ser. No. 10/989,462 filed Nov. 15, 2004, which claims the
benefit of the filing date of U.S. Ser. No. 60/563,514, filed Apr. 19,
2004, and U.S. Ser. No. 60/520,114, filed Nov. 14, 2003. The contents of
these prior applications are hereby incorporated by reference in their
entirety.
TECHNICAL FIELD
[0003]The present invention relates to antibodies that recognize aspartyl (asparaginyl) β-hydroxylase and to methods of using those antibodies to, for example, detect aspartyl (asparaginyl) β-hydroxylase and/or modulate its activity.
BACKGROUND
[0004]Aspartyl(asparaginyl) β-hydroxylase (AAH) catalyzes post-translational hydroxylation of β carbons of specific aspartate and asparagine residues in epidermal growth factor-like domains of numerous proteins, including extracellular matrix proteins, low-density lipoprotein (LDL) receptor, Notch homologs, and Notch ligand homologs (Jia et al., Proc. Natl. Acad. Sci. USA 91(15):7227-7231, 1994; Jia et al., J. Biol. Chem. 267(20):14322-14327, 1992; Gronke et al., Proc Natl Acad Sci USA 86(10):3609-13, 1989). This transmembrane enzyme is a member of the α-ketoglutarate-dependent dioxygenase family of prolyl and lysyl hydroxylases. Overexpression of human AAH (HAAH) has been detected in a number of human cancers, including hepatocellular carcinomas, cholangiocarcinomas, and, neuroectodermal tumors (Lavaissiere et al., J. Clin. Investig. 98:1313-1323, 1996; Sepe et al., Lab. Investig. 82(7):81-891, 2002). The finding that AAH is overexpressed in numerous tumors, and that forced expression increases cell motility and survival indicates that AAH may contribute to malignant transformation in vivo (Sepe et al., Lab. Investig. 82(7):881-891, 2002).
SUMMARY
[0005]This invention is based, in part, on our discovery of human single-chain antibodies that bind aspartyl (asparaginyl) β-hydroxylase (AAH). Accordingly, the invention features antibodies, including complete, multimeric antibodies (e.g., human tetrameric antibodies of the G class (an IgG)) and fragments or other variants thereof that specifically bind an AAH protein. These fragments and variants include single-chain anti-AAH antibodies, fragments or portions of multimeric (e.g., tetrameric) anti-AAH antibodies and other variants that specifically bind an AAH protein. Any of the anti-AAH antibodies or fragments or variants thereof may have been mutagenized by, for example, an affinity maturation process.
[0006]The compositions of the invention include anti-AAH antibodies (e.g., a human monoclonal antibody), fragments or other variants thereof, pharmaceutical compositions containing them, and kits containing them. The antibodies, fragments or other variants thereof may, but do not necessarily, inhibit one or more of AAH's biological activities (e.g., hydroxylation of suitable substrates, whether naturally occurring or non-naturally occurring, in vitro, in cell or tissue culture, or in vivo) and/or one or more of the cellular events mediated by AAH (e.g., cellular growth or proliferation, or cellular motility). As undesirable cellular proliferation and motility occur in connection with pathological conditions in which AAH is overactive or overexpressed (such as cancer), the compositions of the invention can be used to identify patients having such a condition, to assess their prognosis, and/or to treat a patient having, or at risk for developing, such a condition (e.g., cancers or other disorders associated with elevated AAH expression or activity).
[0007]More specifically, the invention features methods of identifying a patient amenable to treatment (e.g., a patient having a cell (or cells) in which AAH is overactive or overexpressed) and therapeutic or prophylactic methods of treating such a patient by administering an effective amount of an anti-AAH antibody (or a fragment or other variant thereof) to the patient (e.g. a human monoclonal antibody or a human single-chain antibody (scFv) that specifically binds HAAH). Also provided are nucleic acids that can be used to express the antibodies of the invention; vectors that include those nucleic acids; cells that contain those nucleic acids or vectors; methods of formulating pharmaceutically acceptable compositions that include an anti-AAH antibody and/or a fragment or other variant thereof; methods of identifying and/or assessing the properties of anti-AAH antibodies (e.g., anti-HAAH antibodies) and/or fragments or other variants thereof; and methods of affinity-maturing anti-AAH antibodies (e.g. human anti-HAAH antibodies) and/or fragments or other variants thereof.
[0008]For ease of reading, we do not repeat the phrase "and/or fragments or other variants thereof" following every occurrence of "antibody" or "antibodies." It is to be understood that wherever an anti-AAH antibody can be used, a fragment or other variant thereof that specifically binds an AAH protein to any useful degree can also be used. We may refer to AAH as "an AAH protein." Unless a contrary meaning is clear, we use the terms "protein," "peptide" and "polypeptide" interchangeably to refer to chains of two or more amino acid residues. Similarly, wherever a nucleic acid encoding an anti-AAH antibody can be used, a nucleic acid encoding a functional fragment or other variant of the anti-AAH antibody can be used; wherever a cell comprising an anti-AAH antibody can be used, one can use a cell comprising a fragment or other variant of an anti-AAH antibody; and so forth. A fragment of an anti-AAH antibody may also be referred to as an "antigen-binding portion" of an anti-AAH antibody, as the fragment may be a portion of an anti-AAH antibody that specifically binds an AAH antigen. While fragments and variants are described further below, we note here that they include Fab, Fab' and F(ab')2 fragments as well as scFvs (e.g., a human scFv that specifically binds HAAH).
[0009]The anti-AAH antibodies or fragments or other variants thereof can consist of, or can include, the amino acid sequences in the Tables of FIGS. 34-38. Alternatively, the anti-AAH antibodies can consist of, or can include, sequences that exhibit a certain degree of identity to the amino acid sequences in the Tables of FIGS. 34-38. For example, the antibodies can include a variable region of the heavy chain (VH) that is at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100% identical) to one of the VH sequences shown in FIGS. 34 and 35. Alternatively, the antibodies can be scFv that are at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to one of the scFvs shown in FIG. 36. Alternatively, the antibodies or fragments or other variants thereof can include complementarity determining regions (CDRs) that are at least 40% identical (e.g., at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 98%) identical to the CDRs shown in FIGS. 37 and 38. Other fragments and variants are described further below.
[0010]The antibody can include a variable light chain (VL or VLC) including a first CDR including (or consisting of) the amino acid sequence Ser-Gln-Ser/Asn-Val-Ser-Ser/His-(Xaa)-Tyr/His-Leu-Ala (SEQ ID NO:320); a second CDR including (or consisting of) the amino acid sequence Asp-Val-Ala-Asn-Xaa-Ala-Ala (SEQ ID NO:321); and a third CDR including (or consisting of) the amino acid sequence Gln-Gln-Arg-Ser-Gln-Trp-Pro-Gln (SEQ ID NO:322). Unless indicated otherwise, in these sequences and those that follow, "Xaa" is any amino acid residue or no amino acid residue. Alternatively, or in addition, the antibody can include a VHC including a first CDR including (or consisting of) the amino acid sequence Tyr/His-Ala-Met-His/Gly (SEQ ID NO:323) and a second CDR including (or consisting of) the amino acid sequence Tyr-Ala-Xaa-Ser-Val-Lys-Gly/Ser (SEQ ID NO:324). In other embodiments, the antibody can include a VLC including a CDR (e.g., a first CDR) including (or consisting of) the amino acid sequence Ser-Gly-Ser-Ser-Ser-Asn-Ile-Gly/Glu-Ser-Asn-His/Tyr-Val-His/Tyr (SEQ ID NO:325). Alternatively, or in addition, the antibody can include a VHC including a CDR (e.g., a first CDR) including (or consisting of) the amino acid sequence Ser/Gly-Asp/Asn-Ser/Gly-Ala-Ala-Trp-Ser/Asn (SEQ ID NO:326) and a second CDR including (or consisting of) the amino acid sequence Arg-Ile/Thr-Tyr/His-Tyr/His-Gly/Arg-Xaa-Lys/Arg-Trp/Arg-Tyr/Arg-- Asn-Asp/Gly-Tyr/His-Ala-Val/Ala-Pro/Ser-Val/Ala-Lys-Ser (SEQ ID NO:327). In other embodiments, the antibody can include a VLC including a CDR (e.g., a second CDR) including (or consisting of) the amino acid sequence Asp-Val-Xaa-Xaa-Arg-Pro-Ser (SEQ ID NO:328). Alternatively, the antibody can include a CDR (e.g., a second CDR) including (or consisting of) the amino acid sequence Leu-Phe/Leu-Ile/Val-His/Tyr-Lys/Arg-Xaa-Asn-Gln-Arg-Pro-Ser (SEQ ID NO:329) and, optionally, a CDR (e.g., a third CDR) including (or consisting of) the amino acid sequence Ala-Trp-Asp-Asp-Ser (SEQ ID NO:330). For example, the third CDR can consist of the sequence Ala-Ala-Trp-Asp-Asp-Ser-Leu-Arg-Gly-Tyr-Val (SEQ ID NO:51). The antibody can also include a VHC including a CDR (e.g., a third CDR) including (or consisting of) the amino acid sequence Ser-Ser-Ser-Trp-Val-Val-Xaa-Phe-Asp/Gly (SEQ ID NO:331) (e.g., Thr-Gly-Tyr-Ser-Ser-Ser-Trp-Val-Val-Asn-Phe-Asp-Tyr (SEQ ID NO:96).
[0011]Any of the antibodies of the invention, regardless of their sequence, can be monoclonal (e.g., monospecific) or polyclonal antibodies; any of the antibodies can be human or humanized; any of the antibodies can be affinity matured; and any of the antibodies can be isolated (e.g., purified to some degree from an animal or cells in which they are produced). Human antibodies include antibodies that have variable and constant regions from human germline immunoglobulin sequences. Such antibodies may have all, or a portion of, a human immunoglobulin heavy chain and all, or a portion of, a human immunoglobulin light chain. This is not to say that the antibodies of the invention (human or non-human) must contain naturally occurring sequences. The antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin nucleic acid sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro). Mutated or affinity matured antibodies are described further below.
[0012]While the antibodies are not limited to those that bind an AAH in a particular manner, the antibodies may bind the catalytic domain of an AAH (e.g., a catalytic domain of HAAH) or they may bind AAH in such a way as to alter the conformation of the catalytic domain or otherwise render it less active. The extent to which the activity of the bound AAH antigen is inhibited can vary. Useful antibodies (or fragments or other variants thereof) within the scope of the present invention include those that inhibit an activity of an AAH (e.g., the catalytic activity of HAAH) to a clinically beneficial degree upon administration to a patient or to an extent that they are useful in in vitro assays. For example, the antibodies can inhibit an enzymatic activity of AAH or HAAH by more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (e.g., about 95%, 98%, or 99%). The inhibition can be assessed relative to a control or a reference sample or standard. Alternatively, an anti-AAH antibody can specifically bind an AAH protein without inhibiting the activity of the AAH protein. Such antibodies are useflI in detecting AAH and can be used to identify a patient having cells in which AAH is overexpressed. The identification process can take place, for example, prior to administering an anti-AAH antibody to the patient that inhibits a biological activity of (e.g., the catalytic activity of) AAH.
[0013]The antibodies of the invention can be further characterized or assessed in terms of their ability to inhibit tumor cell growth or tumor cell motility in cell culture and/or to inhibit tumor cell growth or metastasis in vivo. Affinity can also be measured. For example, an antibody of the invention can have an affinity for an AAH protein (e.g., HAAH) that is equal to or less than about 1 μM. The antibodies of the invention can also be assessed for competition with other antibodies. For example, an antibody of the invention (e.g. a human or humanized antibody) may compete with an anti-AAH murine antibody (e.g., the monoclonal antibody FB50 or the monoclonal antibody 15C7) for binding to an epitope bound by the murine antibody. FB50 and 15C7 are explicitly excluded from the scope of the present invention. An antibody of the invention may also be assessed for its ability to compete with the clone 11 antibody (described herein (see FIG. 34)) for binding to an epitope bound by the clone 11 antibody. Antibodies that effectively compete with the clone 11 antibody are within the scope of the present invention.
[0014]In view of the foregoing, we may use the terms "specific binding" or "specifically binds to" refer to the ability of an antibody to: (1) bind to an AAH with a useful affinity (e.g. an affinity of at least 1×106 M-1, or (2) bind to the AAH with an affinity that is greater than (e.g., at least two-fold greater than) its affinity for a nonspecific antigen, or (3) bind to the AAH with an affinity sufficient to produce a clinically desirable outcome (e.g., an improvement in a sign or symptom of a subject in need of treatment (e.g., a subject who has cancer or other unwanted cellular proliferation)).
[0015]The antibodies can include one or more Fc domains (e.g., an Fc domain of the gamma isotype (e.g., IgG1)). We use the term "isotype" in its conventional sense to refer to the antibody class (e.g., IgM, IgA, IgE, IgD, or IgG1) that is encoded by heavy chain constant region genes. The antibodies of the invention can be of any isotype. Alternatively, or in addition, the antibodies can include a label (e.g., a polypeptide that serves as a marker or reporter sequence or that facilitates purification of the antibody sequence to which it is attached). Suitable labels include a FLAG tag, a histadine tag, or an enzymatically active or fluorescent protein. Alternatively, or in addition, the antibodies can include a toxin.
[0016]In other aspects, the invention features isolated nucleic acid molecules that include a sequence encoding an antibody of the invention (e.g., an anti-AAH scFv); expression vectors (e.g., plasmids) including a nucleic acid sequence encoding an antibody of the invention; and host cells including one or more types of those nucleic acid molecules or expression vectors (e.g., prokaryotic cells or eukaryotic cells such as a yeast or mammalian cells (e.g., Chinese hamster ovary (CHO) cells or tumor cells (e.g., myeloma cells))). Anti-AAH scFvs can be readily converted to multimeric anti-AAH antibodies (e.g., IgGs). More specifically, the invention features antibodies expressed on the surfaces of cells (e.g., displayed on yeast cells). We may use the term "recombinant" to refer to an antibody that is prepared by recombinant means (e.g., an antibody that is expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for a human immunoglobulin gene (or genes), or antibodies prepared by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences). Recombinant antibodies include humanized or CDR grafted antibodies; chimeric antibodies; and antibodies generated in vitro (e.g., by phage display). The antibodies may include constant regions derived from human germline immunoglobulin sequences.
[0017]The invention also features kits containing one or more of the compositions listed above and instructions (regardless of form; whether printed, audio- or visual) for use. For example, the kits of the invention can include an anti-AAH antibody (in a lyophilized or concentrated form or suspended in a physiologically acceptable diluent at a concentration suitable for use (e.g., at a concentration suitable for performing a diagnostic assay or administration to a patient)). The kits can also include nucleic acids, vectors, and/or host cells as described herein. Optionally, any of the kits of the invention can include paraphernalia for administering an anti-AAH antibody (e.g., needles, syringes, alcohol swabs, and bandages) or for using it in a diagnostic assay (e.g., reagents useful as controls).
[0018]The methods of the invention include methods of evaluating AAH expression in a cell. The cell can be a human or non-human cell and/or a cell in which AAH is overactive or overexpressed; the cell can be a cell in vivo or one maintained in tissue culture; the cell can be cancerous (e.g., a tumor cell); and the cell can be obtained from essentially any tissue type (e.g., a cell of the lung, liver, colon, pancreas, prostate, ovary, bile duct, brain, or breast). The cell may or may not be intact (e.g., a tissue homogenate can be used, as can proteins that have been purified from the cell). The methods can include providing an anti-AAH antibody (any of those described herein can be used); contacting the cell (or tissue homogenate or protein purified from the cell) with the antibody under conditions, and for a time, sufficient to allow the antibody to bind AAH expressed by the cell; and detecting the antibody or antigen-binding portion thereof (by, e.g., Western blot analysis, immunohistochemistry, or other antibody-based detection method). The detection step may provide a qualitative or quantitative assessment of AAH expression, and the result can be compared with that obtained from a control sample or with a reference sample or standard. Such methods can be carried out effectively with antibodies that specifically bind AAH, but have no impact on AAH's activity. The methods may be performed as part of an effort to diagnose patients amenable to treatment with the AAH-inhibitory antibodies of the invention.
[0019]The methods of the invention also include methods of modulating (e.g., inhibiting) the activity of an AAH protein in a cell. As with the evaluative methods described above, the cell can be a human or non-human cell and/or a cell in which AAH is overactive or overexpressed; the cell can be a cell in vivo or one maintained in tissue culture; the cell can be cancerous (e.g., a tumor cell); and the cell can be obtained from essentially any tissue type (e.g., a cell of the lung, liver, colon, pancreas, prostate, ovary, bile duct, brain, or breast). The methods can include providing a cell and exposing that cell to an antibody (or fragment or other variant) described herein, for a time, and under conditions sufficient to modulate (e.g., inhibit) AAH activity in the cell. When the cell is exposed to an anti-AAH antibody in vivo, the methods can be described as methods for treating a patient who has a disorder (e.g., a cancer or other disorder of unwanted cellular proliferation) or who is a risk for developing such a disorder (e.g., a patient in which AAH is overactive or overexpressed but who does not yet have a detectable tumor or other sign of cancer). The methods can include administering an antibody (or fragment or other variant thereof) described herein to the patient in an amount and for a time sufficient for the antibody to inhibit proliferation or metastasis of a cancerous cell in the patient. While methods of treatment are described further below, we note that a composition including an anti-AAH antibody can be administered locally (e.g., to the site of a tumor or to tissue remaining after a tumor has been surgically removed) or systemically (e.g., by intravenous injection). The patient can receive a single type of anti-AAH antibody or a combination of antibodies, and the antibody (or antibodies) can be administered in combination with a second agent (e.g. a second chemotherapeutic agent, an analgesic, or anti-emetic).
[0020]Terms relating to treatment refer to the application or administration of a composition of the invention to a patient or to a cell provided from a patient. The composition can be an anti-AAH antibody, a nucleic acid molecule or expression vector encoding same, or a host cell expressing same (any of which can be combined with a physiologically acceptable diluent). The composition can be administered ex vivo to cells isolated from (e.g., removed from) a subject, preferably from the patient in need of treatment. Upon conclusion of the treatment, the cells can be returned to the patient. Moreover, the treatment methods can be prophylactic. For example, they can be applied to a patient who is at risk for developing cancer (there are well established indicators of risk (e.g., levels of cancer-associated antigens, such as PSA and, as noted, AAH per se)). The treatment can be one that cures or heals the patient, but the invention is not so limited. The methods of the invention may also alleviate, relieve, alter, ameliorate, palliate, or improve a sign or symptom of the cancer or the patient's predisposition toward the cancer.
[0021]A "therapeutically effective amount" of an anti-AAH antibody is an amount of an anti-AAH antibody effective to treat a sign or symptom of a disorder (e.g., a cancer, e.g., a tumor or other neoplasm or dysplastic syndrome). A "prophylactically effective amount" of an anti-AAH antibody is an amount of an anti-AAH antibody effective in delaying the occurrence of the onset or recurrence of a disorder (e.g., a cancer), or reducing the severity of a sign or symptom thereof.
[0022]While patients amenable to treatment are described further below, we note here that the patient can have any proliferative disorder associated with overactive or overexpressed AAH. For example, the patient can have a tumor within the lung, liver, colon, pancreas, prostate, ovary, bile duct, brain, or breast containing AAH-positive cells.
[0023]In another aspect, the invention features methods of identifying an antibody that specifically binds to an AAH. The methods can include, for example: (a) providing a library of antibodies (e.g., human antibodies, which may be scFvs); (b) contacting members of the library with AAH proteins or fragments thereof, under conditions that allow the antibodies to bind the polypeptides; and (c) selecting an antibody that binds to the AAH protein (or AAH fragment). The method can further include affinity-maturing the selected antibody. Maturation can be achieved with, for example, methods such as error-prone PCR, or methods in which a nucleic acid encoding the antibody is subjected to recombination with nucleic acid(s) of a library, e.g., using CDR shuffling or chain shuffling techniques. The invention also features methods for making a human monoclonal antibody that specifically binds to an AAH. The methods can include the steps of: identifying an antibody that specifically binds to an AAH protein; expressing a nucleic acid sequence encoding the antibody in a cell; and isolating the expressed antibody from the cell. We may refer to these methods as production methods, and anti-AAH antibodies made by these methods are within the scope of the present invention.
[0024]The antibodies of the invention may be advantageous for various reasons. For example, when less than full-length antibodies are used, the antibody fragments or variants may penetrate tumors more readily. When human or humanized antibodies are administered to human patients, they are unlikely to stimulate an undesirable immune response as potent as that triggered by non-human proteins. The antibodies of the invention are also unlikely to generate other undesirable side effects because HAAH expression is very low or undetectable in non-cancerous tissues, and antibodies directed to HAAH may be less toxic to non-cancerous tissue than less specific treatments. To our knowledge, we have the first human antibodies that bind to HAAH.
[0025]All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026]FIG. 1 is a schematic diagram depicting the fusion proteins displayed on yeast cells for performing screening and binding studies with the yeast surface display technique. Also depicted are various epitope tags and an antigen that can be employed for detection. The fluorescent antigen used in the experiments described herein was a recombinant HAAH protein corresponding to full-length or catalytic domain only, as indicated.
[0027]FIG. 2 is a set of graphs depicting the fluorescence detected by flow cytometry in experiments in which three unique scFv clones expressed on yeast cells were incubated with fluorescent HAAH.
[0028]FIG. 3 is a set of graphs depicting the fluorescence detected by flow cytometry in experiments in which eight unique scFv clones expressed on yeast cells were incubated with a fluorescently labeled fragment of HAAH containing the catalytic domain.
[0029]FIG. 4 is a set of graphs depicting affinity measurements for two anti-HAAH scFv antibody clones.
[0030]FIG. 5 is a set of graphs depicting the fluorescence detected by flow cytometry in experiments in which one scFv clone expressed on yeast cells was incubated with a fluorescently labeled catalytic domain of HAAH or conjugated full-length HAAH. The conjugated full length HAAH proteins were detected using FB50 IgG or 15C7 IgG, as indicated.
[0031]FIG. 6 is a bar graph depicting binding of soluble scFv fragments of 6 unique clones to the catalytic domain of HAAH.
[0032]FIG. 7 is a bar graph depicting binding of soluble scFv fragments of 6 unique clones to full length HAAH.
[0033]FIG. 8 is a set of graphs depicting the fluorescence detected by flow cytometry in experiments in which scFv fragments of three unique clones bound to H640 tumor cells.
[0034]FIG. 9 is a bar graph depicting the percentage of motile H460 tumor cells in the presence of scFv fragments of 5 unique clones, 15C7 (mouse IgG), and in the absence of antibody (first bar).
[0035]FIG. 10 is a graph depicting the fluorescence detected by flow cytometry in an experiment in which affinity matured scFv fragments derived from clone 11 bound to the catalytic domain of HAAH.
[0036]FIG. 11 is a depiction of the amino acid sequence of an affinity-matured clone 11 scFv fragment. Amino acid residues that are changed relative to clone 11 are bold and underlined.
[0037]FIG. 12 is a set of dot-plot graphs depicting two types of fluorescence detected by flow cytometry in experiments in which the original clone 11 scFv fragments, a first round of mutant clone 11 scFv fragments, and a clone derived from the first round of mutants were analyzed. Fluorescence intensity depicted on the X-axis corresponds to the level of scFv fragments displayed on yeast cells. Fluorescence intensity on the Y-axis corresponds to the level of binding to HAAH of the cells.
[0038]FIG. 13 is a set of dot-plot graphs depicting two types of fluorescence detected by flow cytometry in experiments in which the mutant clone 11 scFv fragments were analyzed. Fluorescence intensity depicted on the X-axis corresponds to the level of scFv fragments displayed on yeast cells. Fluorescence intensity on the Y-axis corresponds to the level of binding to HAAH of the cells.
[0039]FIG. 14 is a graph depicting the fluorescence detected by flow cytometry in an experiment in which affinity matured scFv fragments derived from clone 13 bound to the catalytic domain of HAAH.
[0040]FIG. 15 is a depiction of the amino acid sequence of an affinity-matured clone 13 scFv fragment, 13m1. Amino acid residues that are changed relative to clone 13 are bold and underlined.
[0041]FIG. 16 is a set of dot-plot graphs depicting two types of fluorescence detected by flow cytometry in experiments in which the original clone 13 scFv fragments, a first round mutant clone 13 scFv, and a second round mutant clone 13 scFv, were analyzed. Fluorescence intensity depicted on the X-axis corresponds to the level of scFv displayed on yeast cells. Fluorescence intensity on the Y-axis corresponds to the level of binding to HAAH of the cells.
[0042]FIG. 17 is a schematic depiction of a DNA plasmid with restriction enzyme sites for chain shuffling.
[0043]FIG. 18A is a chart listing the VH and VL regions in a wild type clone and five clones generated by chain shuffling from the wild type clone.
[0044]FIG. 18B is a dot-plot graph depicting two types of fluorescence detected by flow cytometry in experiments in which the pool of mutant clones derived from wild type clone 11 by chain shuffling were analyzed. Fluorescence intensity depicted on the X-axis corresponds to the level of scFv fragments displayed on yeast cells. Fluorescence intensity on the Y-axis corresponds to the level of binding to HAAH of the cells.
[0045]FIGS. 19A-K are dot-plot graphs depicting two types of fluorescence detected by flow cytometry in experiments in which the clones produced by chain shuffling were analyzed (FIG. 19A, clone LLm1; FIG. 19B, clone LLm3; FIG. 19C, clone LLm5; FIG. 19D, clone LLm6; FIG. 19E, clone LLm7; FIG. 19F, clone LLm8; FIG. 19G, clone LLm9; FIG. 19H, clone LLm11; FIG. 19I, clone LLm14; FIG. 19J, clone LLm15(3); FIG. 19K, clone LLm20(8)). Fluorescence intensity depicted on the X-axis corresponds to the level of scFv fragments displayed on yeast cells. Fluorescence intensity on the Y-axis corresponds to the level of binding to HAAH of the cells.
[0046]FIG. 20 is a depiction of the amino acid sequences of CDR1, CDR2, and CDR3 regions of a wild-type scFv clone and eleven clones derived by chain shuffling.
[0047]FIG. 21 is a schematic depiction of an acceptor DNA plasmid encoding a wild type clone 11 scFv and a human library insert sequence to be recombined with the plasmid for CDR shuffling.
[0048]FIGS. 22A-D are dot-plot graphs depicting two types of fluorescence detected by flow cytometry in experiments in which the clones produced by chain shuffling were analyzed (FIG. 22A, CM1; FIG. 22B, CM2; FIG. 22C, CM3; FIG. 22D, CM4). Fluorescence intensity depicted on the X-axis corresponds to the level of scFv fragments displayed on yeast cells. Fluorescence intensity on the Y-axis corresponds to the level of binding to HAAH of the cells.
[0049]FIG. 23 is a depiction of the amino acid sequences of CDR1, CDR2, and CDR3 regions of a wild-type scFv clone, and four clones derived by CDR shuffling.
[0050]FIG. 24 is a graph depicting the concentration of selected anti-HAAH scFv fragments vs. anti-HAAH binding activity (as measured by fluorescence).
[0051]FIG. 25 is a schematic diagram depicting the conversion of an scFv fragment to a full length IgG antibody.
[0052]FIGS. 26A-B are graphs depicting the binding of wild-type anti-HAAH IgG antibodies to HAAH as determined by ELISA. The concentration used for each antibody is indicated below the graphs.
[0053]FIG. 27 is a graph depicting the binding of wild-type anti-HAAH IgG antibodies to H460 tumor cells as determined by FACS. The concentrations of antibody used are indicated on the graph.
[0054]FIG. 28 is a graph depicting the binding of selected anti-HAAH IgG mutant antibodies to H460 tumor cells as determined by FACS. The concentrations of antibody used are indicated on the graph.
[0055]FIG. 29A is a graph depicting the concentration of 6-22 IgG antibody vs. binding to H460 cells (as measured by fluorescence).
[0056]FIG. 29B is a graph depicting the concentration of 6-22 IgG antibody vs. binding to FOCUS cells (as measured by fluorescence).
[0057]FIG. 30A is a graph depicting the concentration of CDRm4 IgG antibody vs. binding to H460 cells (as measured by fluorescence).
[0058]FIG. 30B is a graph depicting the concentration of CDRm4 IgG antibody vs. binding to FOCUS cells (as measured by fluorescence).
[0059]FIG. 31 is a graph depicting the binding of scFv 6-22 to H460 cells in the presence or absence of competition from 6-22 IgG antibody.
[0060]FIG. 32A is a graph depicting the binding of HAAH to yeast expressing different scFv fragments in the presence or absence of competition by CDRm4 IgG antibody.
[0061]FIG. 32B is a graph depicting the binding of HAAH to yeast expressing different scFv fragments in the presence or absence of competition by LLm11 IgG antibody.
[0062]FIG. 33A is a graph depicting the binding of HAAH to yeast displaying scFv CDRm4 and the second generation mutant scFv C4m18. Dissociation constants are indicated on the graph.
[0063]FIG. 33B is a graph depicting the display level of first and second generation scFv mutants on yeast.
[0064]FIG. 34 is a Table containing amino acid sequences that can be used as, or that can be included in, anti-AAH antibodies or fragments or other variants thereof.
[0065]FIG. 35 is a Table containing amino acid sequences that can be used as, or that can be included in, anti-AAH antibodies or fragments or other variants thereof (e.g., anti-HAAH affinity-matured, mutagenized antibody regions).
[0066]FIG. 36 is a Table containing amino acid sequences that can be used as, or that can be included in, anti-AAH antibodies or fragments or other variants thereof (e.g., anti-HAAH scFv antibodies).
[0067]FIG. 37 is a Table containing amino acid sequences (anti-AAH antibody CDRs) that can be included in anti-AAH antibodies or fragments or other variants thereof.
[0068]FIG. 38 is a Table containing amino acid sequences (affinity-matured, mutagenized anti-HAAH antibody CDRs) that can be included in anti-AAH antibodies or fragments or other variants thereof.
DETAILED DESCRIPTION
[0069]Aspartyl(asparaginyl) β-hydroxylase (AAH) is a highly conserved alpha-ketoglutarate-dependent dioxygenase that catalyzes post-translational hydroxylation of β carbons of specific aspartate and asparagine residues in epidermal growth factor-like domains of numerous proteins, including extracellular matrix proteins, low-density lipoprotein (LDL) receptor, Notch homologs, and Notch ligand homologs (Jia et al, Proc. Natl. Acad. Sci. USA 91(15):7227-7231, 1994; Jia et al., J. Biol. Chem. 267(20):14322-14327, 1992; Gronke et al., Proc Natl Acad Sci USA 86(10):3609-13, 1989). Overexpression of human AAH (HAAH) has been detected in a number of human cancers, including hepatocellular carcinomas, cholangiocarcinomas, and, neuroectodermal tumors (Lavaissiere et al, J. Clin. Investig. 98:1313-1323, 1996; Sepe et al., Lab. Investig. 82(7):81-891, 2002). The finding that AAH is overexpressed in numerous tumors, and that forced expression increases cell motility and survival indicates that AAH may contribute to malignant transformation in vivo (Sepe et al., Lab. Investig. 82(7):881-891, 2002). Inventions relating to the use of HAAH for diagnosis and treatment of cancer have been described (Radosevich, U.S. Pat. No. 6,166,176; Radosevich, U.S. Pat. No. 6,727,080; Wands et al, U.S. Pat. No. 6,783,758; Wands et al., U.S. Pat. No. 6,797,696; Wands et al, U.S. Pat. No. 6,812,206; Wands et al, U.S. Pat. No. 6,815,415).
[0070]The present invention relates to antibodies and antigen binding portions thereof having binding specificity for AAH or a portion of AAH. In particular, the invention relates to human monoclonal antibodies that specifically bind to AAH. In one embodiment, the antibodies or antigen binding portions thereof have specificity for human AAH (HAAH). Antibodies that inhibit one or more functions characteristic of a AAH are within the scope of the present invention, whether that function is an enzymatic activity (e.g., hydroxylase activity) or a function manifested on the cellular level (e.g., facilitating motility of a tumor cell). Thus, for example, an anti-AAH antibody can be one that inhibits motility of a tumor cell. Alternatively, or in addition, an antibody of the invention may inhibit (reduce or prevent) the interaction of AAH with a natural ligand, such as a protein containing an EGF-like domain (e.g., an extracellular matrix protein). Human monoclonal antibodies directed against (e.g., antibodies that specifically bind) AAH can inhibit functions mediated by AAH, including modulation of substrate activity by hydroxylation. Preferably, the antibodies and antigen binding portions thereof can bind AAH with an affinity of greater than 1×106 M-1.
[0071]The HAAH amino acid sequence is found in GenBank® under accession number I38423 (GI:7433245). One of ordinary skill in the art can readily retrieve the sequence from GenBank® or another source. The transmembrane domain of HAAH is designated as being between amino acids 341-374 of the GenBank® sequence. The extracellular (or luminal) portion of the molecule corresponds to the C-terminal end. Anti-AAH antibodies, whether full-length or not, will interact with (e.g., bind to) AAH or a fragment of that protein (e.g. an anti-HAAH antibody will bind to HAAH). The antibody may bind to an epitope of AAH (e.g., a conformational or a linear epitope) or to a fragment of the full-length AAH protein. Conformational epitopes are typically lost when exposed to a denaturing solvent.
[0072]Antibodies: Antibodies of the invention can assume various configurations. For example, the antibody can be a tetramer (e.g. an antibody having two heavy chains and two light chains) or a single-chain antibody. Accordingly, the antibodies of the invention include proteins that may have one or two heavy (H) chain variable regions, and one or two light chain variable regions. The VHC and VLC regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with regions that are more conserved, termed "framework regions" (FRs). The extent of the FRs and CDRs has been defined (see, Kabat, E. A., et al. Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, et al., J. Mol. Biol. 196:901-917, 1987, which are incorporated herein by reference). Where an antibody of the invention includes one or more VHCs and/or one or more VLCs, each VHC and VLC can be composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0073]The VHC or VLC chain of an antibody of the invention can further include all or part of a heavy or light chain constant region. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region includes three domains: CH1, CH2 and CH3. The light chain constant region is comprised of one domain: CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof (e.g., IgG1, IgG2, IgG3, and IgG4)), wherein the light chains of the immunoglobulin may be of types kappa or lambda.
[0074]Antibodies may also be referred to as "immunoglobulins" (proteins consisting of one or more polypeptides substantially encoded by immunoglobulin genes, the anti-AAH antibodies of the invention may also be referred to as anti-AAH immunoglobulins, and may contain sequences encoded by one or more of the human immunoglobulin genes). The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 kDa and 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin heavy chains (about 50 kDa and 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). The antibodies or immunoglobulins of the present invention may include CDRs (which are described further herein) from a human or non-human source. The framework of the immunoglobulin can be human, humanized, or non-human, e.g., a murine framework modified to decrease antigenicity in humans, or a synthetic framework, e.g., a consensus sequence.
[0075]The term "antigen-binding portion" of an antibody (or simply "antibody portion," or "portion"), as used herein, refers to a portion of an antibody that specifically binds to an AAH (e.g., HAAH), e.g., a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to an AAH. Examples of binding portions encompassed within the term "fragment (or antigen-binding portion) or other variant thereof" of an antibody include (i) an Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHC and CH1 domains; (iv) a Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VHC domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to specifically bind, e.g., an antigen binding portion of a variable region. An antigen-binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. Science 242:423-426, 1988; and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody portions are obtained using conventional techniques known to those with skill in the art, and the portions are screened for utility in the same manner as are intact antibodies. An Fab fragment can result from cleavage of a tetrameric antibody with papain; Fab' and F(ab')2 fragments can be generated by cleavage with pepsin.
[0076]As used herein, the term "human antibody" includes any antibody in which the framework residues correspond to human germline sequences and the CDRs result from V(D)J recombination and somatic mutations. However, human antibodies may also comprise amino acid residues not encoded in human germline immunoglobulin nucleic acid sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro). It has been demonstrated that in vivo somatic mutation of human variable genes results in mutation of framework residues (see Nat. Immunol. 2:537, 2001). Such an antibody would be termed "human" given its source, despite the framework mutations. Mouse antibody variable domains also contain somatic mutations in framework residues (See Sem. Immunol 8:159, 1996). Consequently, transgenic mice containing the human Ig locus produce antibodies that are commonly referred to as "fully human," even though they possess an average of 4.5 framework mutations (a range of 1-8 in this work: Nat Genet. February 1997;15(2):146-56). Accepted usage therefore indicates that an antibody variable domain gene based on germline sequence but possessing framework mutations introduced by, for example, an in vivo somatic mutational process is termed "human." Thus, the invention encompasses human antibodies that specifically bind AAH (e.g., HAAH, even where those antibodies include mutations (e.g., mutations within the FR) and fragments or other variants thereof (e.g., single chain antibodies that include the VLC and VHC of a multimeric human antibody). For example, the human antibodies of the invention can have 1-8 framework mutation (e.g., about 2, 4, 6, or 8 substitutions, additions, or deletions). Preferably, the sequence of the original human antibody is a human germline sequence.
[0077]Human single-chain antibodies specific for HAAH were produced as described herein. In a particular embodiment, the invention provides antibodies that have specificity for HAAH, and bind to an epitope bound by an antibody described herein (e.g., the antibody encoded by clone 11 or an affinity-matured derivative of clone 11). Antibodies that bind an epitope that overlaps with an epitope bound be an antibody described herein can be identified by their ability to compete with an EGF-like domain for binding to HAAH (e.g., to cells bearing HAAH, such as HAAH transfectants, or H460 tumor cells). The binding site of an anti-AAH antibody can be within the catalytic domain of HAAH.
[0078]The anti-AAH antibodies can be polyclonal or monoclonal. The antibodies and antigen binding portions thereof described herein are useful in therapeutic compositions and regimens, diagnostic compositions and regimens, and in assays requiring an agent that can identify or inhibit an AAH protein. The present invention encompasses an antibody or antigen binding portion thereof for use in therapy (including prophylaxis) or diagnosis (e.g., of particular diseases or conditions such as cancers), and use of such antibodies or antigen binding portions thereof for the manufacture of a medicament for use in treatment of diseases or conditions as described herein.
[0079]Single chain antibodies, and chimeric, humanized or CDR-grafted antibodies, as well as chimeric or CDR-grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present invention and the term "antibody." The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as contiguous polypeptides using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous polypeptide. See, e.g., Cabilly et al, U.S. Pat. No. 4,816,567; Cabilly et al, European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No.4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al, WO 86/01533; Neuberger, M. S. et al, European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; and Winter, European Patent No. 0,239,400 B1. See also, Newman et al, BioTechnology 10: 1455-1460,1992, regarding CDR-graft antibody, and Ladner et al, U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science 242: 423-426,1988 regarding single chain antibodies.
[0080]In addition, antigen binding portions of antibodies, including fragments of chimeric, humanized, CDR-grafted or single chain antibodies, can also be produced and are within the scope of the present invention. Antigen binding portions of the antibodies retain at least one binding function of the full-length antibody from which they are derived. Preferred antigen binding portions retain an antigen binding function of a corresponding full-length antibody (e.g., specificity for an AAH). Functional fragments can retain the ability of the full-length antibody to inhibit one or more functions characteristic of a AAH, such as AAH's hydroxylase activity. For example, a functional fragment can inhibit hydroxylation of an EGF-like domain. These EGF-like domains contain conserved motifs that form repetitive sequences in diverse proteins, such as clotting factors, extracellular matrix proteins, low-density lipoprotein receptor, Notch homologues or Notch ligand homologues. Any AAH substrate, including those just described, can be used in assays to assess an anti-AAH antibody. Exemplary AAH assay substrates include EGF-IX1H (Gronke et al., Proc Natl Acad Sci USA 86(10):3609-13, 1989), EGF-X1H (Gronke et al., J. Biol. Chem. 265:8558-8565,1990), and EGF-Asn (Gronke et al, J. Biol. Chem. 265:8558-8565,1990; Wang et al., J. Biol. Chem. 266:14004-14010,1991).
[0081]For example, antibody portions capable of binding to a AAH or a fragment thereof include Fv, Fab, Fab' and F(ab')2 fragments. Such portions can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain.
[0082]The invention provides chimeric antibodies that can be prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain). One example of a chimeric antibody of the present invention is an antibody containing one or more antibody chains comprising a CDR (e.g., one or more CDRs of an antibody described herein) and a framework region derived from a light and/or heavy chain of a second antibody (e.g., of human origin; e.g., CDR-grafted antibodies with or without framework changes). In one embodiment, the chimeric antibody can compete with the murine 15C7 or FB50 monoclonal antibody for binding to HAAH. The antigen binding region of the chimeric antibody can be derived from an antibody clone described herein (e.g., clone 11, or a mutant of clone 11; e.g., as in a chimeric antibody comprising CDR1, CDR2 and CDR3 of the clone 11 light chain and CDR1, CDR2 and CDR3 of the clone 11 heavy chain). Chimeric or CDR-grafted single chain antibodies also include humanized immunoglobulin. See, e.g., Cabilly et al, U.S. Pat. No. 4,816,567; Cabilly et al, European Patent No. 0,125,023 B1; Queen et al, European Patent No. 0,451,216 B1; Boss et al, U.S. Pat. No. 4,816,397; Boss et al, European Patent No. 0,120,694 B1; Neuberger et al, WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E. A. et al, European Patent Application No. 0,519,596 A1. See also, Ladner et al, U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird et al, Science 242: 423-426,1988), regarding single chain antibodies.
[0083]Chimeric antibodies can be produced using synthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired chimeric chain. For example, nucleic acid (e.g., DNA) sequences coding for variable regions can be constructed using PCR mutagenesis methods to alter DNA sequences encoding an antibody chain, e.g., using methods employed to generate humanized antibodies (see e.g., Kanunan, et al, Nucl. Acids Res. 17: 5404,1989; Sato, et al, Cancer Research 53:851-856,1993; Daugherty, et al, Nucleic Acids Res. 19(9): 2471-2476,1991; and Lewis and Crowe, Gene 101: 297-302,1991). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al, U.S. Pat. No. 5,514,548; Hoogenboom et al, WO 93/06213, published Apr. 1, 1993)).
[0084]Other suitable methods of producing or isolating anti-AAH antibodies include, for example, methods that rely upon immunization of transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies (see e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA 90: 2551-2555, 1993; Jakobovits et al, Nature 362: 255-258, 1993; Lonberg et al, U.S. Pat. No. 5,545,806; Surani et al., U.S. Pat. No. 5,545,807).
[0085]Antibodies that specifically bind to AAH can be identified by expressing recombinant antibodies in a library and selecting members of the library that bind AAH. The affinity of the selected antibodies for AAH can be further enhanced by affinity-maturing these antibodies, e.g., using PCR mutagenesis, chain shuffling, or CDR shuffling techniques in conjunction with one or more cycles of screening, as described herein. Other methods can also be used to generate anti-AAH antibodies. For example, anti-AAH antibodies can be produced by immunizing animals. A variety of methods have been described for preparing antigen for immunization and for generating monoclonal antibodies from immunized animals (see e.g., Kohler et al, Nature 256:495-497, 1975; Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976; Milstein et al., Nature 266:550-552,1977; Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)). Generally, a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line) with antibody producing cells. The antibody producing cell, preferably those of the spleen or lymph nodes, are obtained from immunized animals. The fused cells (hybridomas) can be isolated using selective culture conditions and cloned by limiting dilution. Cells that produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
[0086]The antibody or an antigen-binding portion thereof can include, for example, a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to one of the following sequences: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:119, SEQ ID NO:121; SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260; SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, and SEQ ID NO:270.
[0087]The antibody or an antigen-binding portion thereof can include, for example, a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to one of the following sequences: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261; SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, and SEQ ID NO:271.
[0088]The antibody or an antigen-binding portion thereof can include, for example, a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:1 and a variable light chain region at least 80% identical to (e.g., 85%, 90%, 95%, 98% or 100%) SEQ ID NO:2; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:3 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:4; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:5 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:6; a variable heavy chain region at least 80% identical to (e.g., 85%, 90%, 95%, 98% or 100%) SEQ ID NO:7 and a variable light chain region at least 80% identical(e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:8; a variable heavy chain region at least 80% identical (e.g., 85%,90%,95%,98% or 100%) to SEQ ID NO:9 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:10; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:11 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQID NO:12; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:13 and avariable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:14; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:15 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:16; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:17 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ IDNO:18; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:19 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:20; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:21 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:22; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:23 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:24; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:25 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:26; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:27 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:28; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:29 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:30; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:119 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:120; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:121 and a variable light chain region at least 80% identical (e.g. 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:122; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:123 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:124; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:125 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:126; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:127 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:128; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:129 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:130; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:131 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:132; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:133 and avariable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:134; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:135 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:136; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:137 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:138; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:139 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:140; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:141 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:142; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:143 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:144; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:145 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:146; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:147 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:148; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:254 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:255; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:256 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:257; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:258 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:259; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:260 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:261; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:262 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:263; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:264 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:265; a variable heavy chain region at least 80% identical (e.g. 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:266 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:267; a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:268 and a variable light chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:269; or a variable heavy chain region at least 80% identical (e.g., 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:270 and a variable light chain region at least 80% identical (e.g. 85%, 90%, 95%, 98% or 100%) to SEQ ID NO:271.
[0089]In various embodiments, the antibody or antigen-binding portion thereof includes a complementarity determining region (CDR) that is at least 40% identical (e.g., 40%, 60%, 80%, 85%, 90%, 95%, 98% or 100%) to any one of SEQ ID NOs. 46-81 or 281-313.
[0090]In various embodiments, the antibody or fragment or other variant thereof includes a complementarity determining region (CDR) that is at least 40% identical (e.g., 40%, 60%, 80%, 85%, 90%, 95%, 98% or 100%) to any one of SEQ ID NOs. 46-51, 54, 56, 82-117, 164-250, or 314-319. The antibody or fragment or other variant thereof can also be, or can include: a variable heavy chain region at least 80% identical to SEQ ID NO:1 and a variable light chain region at least 80% identical to SEQ ID NO:2; a variable heavy chain region at least 80% identical to SEQ ID NO:3 and a variable light chain region at least 80% identical to SEQ ID NO:4; a variable heavy chain region at least 80% identical to SEQ ID NO:5 and a variable light chain region at least 80% identical to SEQ ID NO:6; a variable heavy chain region at least 80% identical to SEQ ID NO:7 and a variable light chain region at least 80% identical to SEQ ID NO:8; a variable heavy chain region at least 80% identical to SEQ ID NO:9 and a variable light chain region at least 80% identical to SEQ ID NO:10; a variable heavy chain region at least 80% identical to SEQ ID NO:11 and a variable light chain region at least 80% identical to SEQ ID NO:12; a variable heavy chain region at least 80% identical to SEQ ID NO:13 and a variable light chain region at least 80% identical to SEQ ID NO:14; a variable heavy chain region at least 80% identical to SEQ ID NO:15 and a variable light chain region at least 80% identical to SEQ ID NO:16; a variable heavy chain region at least 80% identical to SEQ ID NO:17 and a variable light chain region at least 80% identical to SEQ ID NO:18; a variable heavy chain region at least 80% identical to SEQ ID NO:19 and a variable light chain region at least 80% identical to SEQ ID NO:20; a variable heavy chain region at least 80% identical to SEQ ID NO:21 and a variable light chain region at least 80% identical to SEQ ID NO:22; a variable heavy chain region at least 80% identical to SEQ ID NO:23 and a variable light chain region at least 80% identical to SEQ ID NO:24; a variable heavy chain region at least 80% identical to SEQ ID NO:25 and a variable light chain region at least 80% identical to SEQ ID NO:26; a variable heavy chain region at least 80% identical to SEQ ID NO:27 and a variable light chain region at least 80% identical to SEQ ID NO:28; a variable heavy chain region at least 80% identical to SEQ ID NO:29 and a variable light chain region at least 80% identical to SEQ ID NO:30; a variable heavy chain region at least 80% identical to SEQ ID NO:119 and a variable light chain region at least 80% identical to SEQ ID NO:120; avariable heavy chain region at least 80% identical to SEQ ID NO:121 and a variable light chain region at least 80% identical to SEQ ID NO:122; a variable heavy chain region at least 80% identical to SEQ ID NO:123 and a variable light chain region at least 80% identical to SEQ ID NO:124; a variable heavy chain region at least 80% identical to SEQ ID NO:125 and a variable light chain region at least 80% identical to SEQ ID NO:126; a variable heavy chain region at least 80% identical to SEQ ID NO:127 and a variable light chain region at least 80% identical to SEQ ID NO:128; a variable heavy chain region at least 80% identical to SEQ ID NO:129 and a variable light chain region at least 80% identical to SEQ ID NO:130; a variable heavy chain region at least 80% identical to SEQ ID NO:131 and a variable light chain region at least 80% identical to SEQ ID NO:132; a variable heavy chain region at least 80% identical to SEQ ID NO:133 and a variable light chain region at least 80% identical to SEQ ID NO:134; a variableheavy chain region at least 80% identical to SEQ ID NO:135 and avariable light chain region at least 80% identical to SEQ ID NO:136; a variable heavy chain region at least 80% identical to SEQ ID NO:137 and a variable light chain region at least 80% identical to SEQ ID NO:138; a variable heavy chain region at least 80% identical to SEQ ID NO:139 and a variable light chain region at least 80% identical to SEQ ID NO:140; a variable heavy chain region at least 80% identical to SEQ ID NO:141 and a variable light chain region at least 80% identical to SEQ ID NO:142; a variable heavy chain region at least 80% identical to SEQ ID NO:143 and a variable light chain region at least 80% identical to SEQ ID NO:144; a variable heavy chain region at least 80% identical to SEQ ID NO:145 and a variable light chain region at least 80% identical to SEQ ID NO:146; a variable heavy chain region at least 80% identical to SEQ ID NO:147 and a variable light chain region at least 80% identical to SEQ ID NO:148; a variable heavy chain region at least 80% identical to SEQ ID NO:254 and a variable light chain region at least 80% identical to SEQ ID NO:255; a variable heavy chain region at least 80% identical to SEQ ID NO:256 and a variable light chain region at least 80% identical to SEQ ID NO:257; a variable heavy chain region at least 80% identical to SEQ ID NO:258 and a variable light chain region at least 80% identical to SEQ ID NO:259; a variable heavy chain region at least 80% identical to SEQ ID NO:260 and a variable light chain region at least 80% identical to SEQ ID NO:261; a variable heavy chain region at least 80% identical to SEQ ID NO:262 and a variable light chain region at least 80% identical to SEQ ID NO:263; a variable heavy chain region at least 80% identical to SEQ ID NO:264 and a variable light chain region at least 80% identical to SEQ ID NO:265; a variable heavy chain region at least 80% identical to SEQ ID NO:266 and a variable light chain region at least 80% identical to SEQ ID NO:267; a variable heavy chain region at least 80% identical to SEQ ID NO:268 and a variable light chain region at least 80% identical to SEQ ID NO:269; or a variable heavy chain region at least 80% identical to SEQ ID NO:270 and a variable light chain region at least 80% identical to SEQ ID NO:271.
[0091]As used herein, the term "substantially identical" (or "substantially homologous") refers to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities. In the case of antibodies, the second antibody has the same specificity and has at least 50% of the affinity of the first antibody.
[0092]Calculations of "homology" or "identity" between two sequences can be performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In different embodiments, the length of a reference sequence aligned for comparison purposes is at least 50% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
[0093]The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. The percent homology between two amino acid sequences is determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, 1970, algorithm which has been incorporated into the GAP program in the GCG software package, using a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0094]As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which is incorporated herein by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.
[0095]It is understood that the antibodies and antigen binding portions thereof of the invention may have additional conservative or non-essential amino acid substitutions (a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide, such as a binding agent, e.g., an antibody, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change).
[0096]Whether or not a particular substitution will be tolerated, i.e., will not adversely affect desired biological properties, such as binding activity, can be determined as described in Bowie et al., Science, 247:1306-1310, 1990. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).
[0097]As described herein, antibodies of the present invention can inhibit hydroxylase activity of AAH and/or inhibit a function associated with hydroxylase activity, such as cell motility. As discussed below, various methods can be used to assess inhibition of AAH activity and/or function associated with the activity.
Binding Assays
[0098]As used herein "mammalian AAH protein" refers to naturally occurring or endogenous mammalian AAH proteins and to proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous corresponding mammalian AAH protein (e.g., recombinant proteins). Accordingly, as defined herein, the term includes polymorphic or allelic variants, and other isoforms of a mammalian AAH (e.g. produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e.g. glycosylated, unglycosylated). AAH proteins can be isolated and/or recombinant proteins (including synthetically produced proteins). Naturally occurring or endogenous mammalian AAH can be recovered or isolated from a source which naturally produces AAH, for example, a tumor cell.
[0099]"Functional variants" of AAH proteins include functional fragments, functional mutant proteins, and/or functional fusion proteins (e.g., produced via mutagenesis and/or recombinant techniques). Generally, fragments or portions of AAH proteins include those having a deletion (i.e., one or more deletions) of an amino acid (i.e., one or more amino acids) relative to the mature mammalian AAH (such as N-terminal, C-terminal or internal deletions).
[0100]Generally, mutants of AAH proteins include natural or artificial variants of an AAH protein differing by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues. Such mutations can be in a conserved region or nonconserved region, extracellular, cytoplasmic, or transmembrane region, for example.
[0101]A "functional fragment or portion", "functional mutant" and/or "functional fusion protein" of an AAH protein refers to an isolated and/or recombinant protein or polypeptide which has at least one function characteristic of a AAH protein as described herein, such as a hydroxylase activity.
[0102]A composition comprising an isolated and/or recombinant mammalian AAH or portion thereof can be maintained under conditions suitable for binding, the receptor is contacted with an antibody to be tested, and binding is detected or measured. In one embodiment, a receptor protein can be expressed in cells which express AAH or in cells stably or transiently transfected with a construct comprising a nucleic acid sequence which encodes an AAH or portion thereof. The cells are maintained under conditions appropriate for expression of receptor. The cells are contacted with an antibody under conditions suitable for binding (e.g., in a suitable binding buffer), and binding is detected by standard techniques. To measure binding, the extent of binding can be determined relative to a suitable control (e.g., compared with background determined in the absence of antibody, compared with binding of a second antibody (i.e., a standard), compared with binding of antibody to untransfected cells). A cellular fraction, such as a membrane fraction, containing AAH can be used in lieu of whole cells.
[0103]In one embodiment, the antibody is labeled with a suitable label (e.g. fluorescent label, isotope label, enzyme label), and binding is determined by detection of the label. In another embodiment, bound antibody can be detected by labeled second antibody. Specificity of binding can be assessed by competition or displacement, for example, using unlabeled antibody or a ligand as competitor.
[0104]Binding inhibition assays can also be used to identify antibodies that bind AAH and inhibit binding of another compound such as an EGF-like domain. For example, a binding assay can be conducted in which a reduction in the binding of an EGF-like domain (in the absence of an antibody), as compared to binding of the ligand in the presence of the antibody, is detected or measured. AAH can be contacted with a protein containing an EGF-like domain and antibody simultaneously, or one after the other, in either order. A reduction in the extent of binding of the protein in the presence of the antibody, is indicative of inhibition of binding by the antibody. For example, binding of the EGF-like domain could be decreased or abolished.
[0105]Other methods of identifying the presence of an antibody which binds AAH are available, such as other suitable binding assays, or methods which monitor events which are triggered by AAH activity, e.g., cellular transformation, or cell motility.
[0106]It will be understood that the inhibitory effect of antibodies of the present invention can be assessed in a binding inhibition assay. Competition between antibodies for binding can also be assessed in the method. Antibodies which are identified in this manner can be further assessed to determine whether, subsequent to binding, they act to inhibit other functions of AAH and/or to assess their therapeutic utility.
Assays to Determine Antibody Activity
[0107]AAH hydroxylates the β carbon of aspartic acid or asparagines residues in epidermal growth factor (EGF)-like domains of proteins in the presence of ferrous iron. EGF-like domains, which contain a conserved CX7CX4CX10CXCX8C sequence, are present in many diverse proteins, such as clotting factors, extracellular matrix proteins, low density lipoprotein receptor, Notch homologs, and Notch ligand homologs. Hydroxylation of AAH substrates can involve direct detection of hydroxylase activity, or can be measured by indirectly, e.g., using assays that detect a biological activity downstream of hydroxylation.
[0108]An assay to determine whether a human anti-AAH antibody inhibits AAH activity can be performed by comparing the level of hydroxylation in an enzymatic reaction in which the test antibody is present compared to a parallel reaction in the absence of a compound, or compared to a predetermined control value. Standard hydroxylase assays are known. See, e.g., Lavaissiere et al, J. Clin. Invest. 98:1313-1323,1996; Jia et al., J. Biol. Chem. 267:14322-14327,1992; Wang et al., J. Biol. Chem. 266:14004-14010,1991; or Gronke et al., J. Biol. Chem. 265:8558-8565,1990. Hydroxylase activity can be measured using carbon dioxide (14CO2 capture assay) in a microtiter plate (Zhang et al, Anal Biochem. 271:137-142, 1999).
[0109]Modulation of AAH activity can be determined by examining biochemical effects downstream of hydroxylation, including effects on specific substrates, and effects on cellular processes. AAH activity increases cell motility, proliferation, survival, and cell cycle progression. Inhibition of AAH activity by an antibody can be determined by detecting a reduction in one of these processes in the presence of the antibody.
[0110]Modulation of cell motility can be assayed, e.g., using a motility assay. Generally, motility assays monitor the directional movement or migration of a suitable cell (such as a tumor cell) into or through a barrier (e.g., a filter), toward increased levels of a compound (e.g., a growth factor or other polypeptide), from a first surface of the barrier toward an opposite second surface. Membranes or filters provide convenient barriers, such that the directional movement or migration of a suitable cell into or through a filter, toward increased levels of a compound, from a first surface of the filter toward an opposite second surface of the filter, is monitored. One can detect or measure inhibition of the migration of cells in a suitable container from a first chamber into or through a microporous membrane into a second chamber which contains an antibody to be tested, and which is divided from the first chamber by the membrane. A suitable membrane, having a suitable pore size for monitoring specific migration in response to compound, including, for example, nitrocellulose, polycarbonate, is selected. For example, pore sizes of about 3-8 microns can be used.
[0111]To assess migration and inhibition of migration, the number of cells crossing the filter that remain adherent to the second surface of the filter, and/or the number of cells that accumulate in the second chamber can be determined using standard techniques (e.g., microscopy). In one embodiment, the cells are labeled with a detectable label (e.g., radioisotope, fluorescent label, antigen or epitope label), and migration can be assessed in the presence and absence of the antibody by determining the presence of the label adherent to the membrane and/or present in the second chamber using an appropriate method (e.g., by detecting radioactivity, fluorescence, immunoassay). The extent of migration induced by an antibody agonist can be determined relative to a suitable control (e.g., compared to background migration determined in the absence of the antibody, compared to the extent of migration induced by a second compound (i.e., a standard), compared with migration of untransfected cells induced by the antibody). A reduction in the extent of migration induced by the compound (e.g., calf serum) in the presence of the antibody is indicative of inhibitory activity.
[0112]In one embodiment, cells are placed in the upper chamber of a Boyden chamber-type culture insert in serum-free medium. Medium supplemented with 1%-2% fetal calf serum is placed in the lower chamber to provide a stimulus for migration. Cells are incubated for approximately 4 hours to allow migration to occur. Cell numbers in the upper and lower chambers are quantified. Viable cells in each chamber can be quantified using an ATP monitoring system such as ATPLite® (Perkin Elmer®; see, e.g., Sepe, et al. Lab. Invest. 82:881-891, 2002 for a description of a motility assay).
[0113]AAH overexpression is linked to cellular proliferation and malignant transformation. Inhibition of AAH activity can be assayed by measuring cellular characteristics of malignant phenotypes, such as transformation, anchorage-independent cell growth, and tumorigenicity in nude mice. Transformation can be assessed by transfecting NIH 3T3 cells with AAH and observing the number of transformed foci (Copeland and Cooper, Cell 16(2):347-56, 1979). Anchorage-independent cell growth can be assayed by transfecting cells with AAH, isolating transfectants, and suspending transfectants in complete medium containing 0.4% low-melting agarose laid over a bottom layer of medium containing 0.53% low-melting agarose.
[0114]To assay tumorigenicity in vivo, AAH-transfected clones (or AAH-expressing tumor cells) are injected into nude mice. In a typical assay, approximately 1-10 million cells are injected subcutaneously. After growth for 1 week-1 month, animals are sacrificed, and tumors are removed and weighed. Antibodies can be tested by injection into animals implanted with AAH-transfectants or tumor cells and comparing growth of the implanted cells to growth of cells in animals receiving a control injection (e.g., saline, or a non-specific antibody).
[0115]Antibodies with inhibitory activity may reduce efficiency of cell growth, transformation, and tumorigenicity in these assays.
Diagnostic and Therapeutic Applications
[0116]The antibodies of the present invention are useful in a variety of applications, including research, diagnostic and therapeutic applications. In one embodiment, the antibodies are labeled with a suitable label (e.g., fluorescent label, chemiluminescent label, isotope label, epitope or enzyme label).
[0117]Overexpression of HAAH is associated with malignant transformation. Antibodies or antigen binding portions thereof that block and/or inhibit the activity of HAAH can be used to inhibit cell transformation and/or to diagnose transformed cells. Accordingly, the present invention provides a method of inhibiting AAH activity of a cell which expresses an AAH or portion thereof, comprising contacting the cell with an effective amount of an antibody or antigen binding portion thereof which binds to the AAH or a portion of the AAH. The cell can be a cell of a subject (e.g., a tumor cell), and the antibody or antigen binding portion thereof can be administered to the subject in vivo. Therapeutic use of an antibody or antigen binding portion thereof includes prophylactic use (e.g., for treatment of a patient who may be at risk for developing a cancer).
[0118]The anti-AAH antibody or antigen binding portion thereof can be administered in combination with one or more other therapeutic agents such as an anti-cancer agent. Nonlimiting examples of anti-cancer agents include, e.g., antimicrotubule agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis (including cell death genes), radioactive compounds, and antibodies against other tumor-associated antigens (including naked antibodies, immunotoxins and radioconjugates). Examples of the particular classes of anti-cancer agents are provided in detail as follows: antitubulin/antimicrotubule, e.g., paclitaxel, vincristine, vinblastine, vindesine, vinorelbin, taxotere; topoisomerase I inhibitors, e.g., topotecan, camptothecin, doxorubicin, etoposide, mitoxantrone, daunorubicin, idarubicin, teniposide, amsacrine, epirubicin, merbarone, piroxantrone hydrochloride; antimetabolites, e.g., 5-fluorouracil (5-FU), methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, cytarabine/Ara-C, trimetrexate, gemcitabine, acivicin, alanosine, pyrazofurin, N-Phosphoracetyl-L-Asparate (PALA), pentostatin, 5-azacitidine, 5-Aza 2'-deoxycytidine, ara-A, cladribine, 5-fluorouridine, FUDR, tiazofurin, N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-- 2-thenoyl]-L-glutamic acid; alkylating agents, e.g., cisplatin, carboplatin, mitomycin C, melphalan, thiotepa, busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen mustard, uracil mustard, pipobroman, 4-ipomeanol; agents acting via other mechanisms of action, e.g., dihydrolenperone, spiromustine, and desipeptide; biological response modifiers, e.g., to enhance anti-tumor responses, such as interferon; apoptotic agents, such as actinomycin D; and anti-hormones, for example anti-estrogens such as tamoxifen or, for example antiandrogens such as 4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluorometh- yl)propionanilide.
[0119]The anti-AAH antibodies of the present invention also have value in diagnostic applications. An anti-AAH antibody can be used to monitor growth and/or metastasis of a tumor in vivo, and may be used as a diagnostic indicator of disease stage. Human antibodies are not immunogenic in humans, and therefore human antibodies may be more appropriate for in vivo diagnostic applications than mouse antibodies.
[0120]For diagnostic purposes, the antibodies or antigen binding portions can be labeled or unlabeled. Typically, diagnostic assays entail detecting the formation of a complex resulting from the binding of an antibody or portion to AAH. The antibodies or portions can be directly labeled. A variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens, and the like). Numerous appropriate immunoassays are known to the skilled artisan (see, for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654 and 4,098,876). Unlabeled antibodies or fragments can also be used in combination with another (i.e., one or more) suitable reagent that can be used to detect antibody, such as a labeled antibody (e.g., a second antibody) reactive with the first antibody (e.g., anti-idiotype antibodies or other antibodies that are specific for the unlabeled immunoglobulin) or other suitable reagent (e.g., labeled protein A).
[0121]Kits for use in detecting the presence of AAH in a biological sample can also be prepared. Such kits will include an antibody or antigen binding portion thereof which binds to an AAH protein or portion of said receptor, as well as one or more ancillary reagents suitable for detecting the presence of a complex between the antibody or portion and AAH or portion thereof. The antibody compositions of the present invention can be provided in lyophilized form, either alone or in combination with additional antibodies specific for other epitopes. The antibodies, whether labeled or unlabeled, can be included in the kits with adjunct ingredients (e.g., buffers, such as Tris, phosphate and carbonate, stabilizers, excipients, biocides and/or inert proteins, e.g., bovine serum albumin). For example, the antibodies can be provided as a lyophilized mixture with the adjunct ingredients, or the adjunct ingredients can be separately provided for combination by the user. Where a second antibody capable of binding to the monoclonal antibody is employed, such antibody can be provided in the kit, for instance in a separate vial or container. The second antibody, if present, is typically labeled, and can be formulated in an analogous manner with the antibody formulations described above.
[0122]Similarly, the present invention also relates to a method of detecting and/or quantifying expression of AAH or portion of the enzyme by a cell, in which a composition comprising a cell or fraction thereof (e.g., membrane fraction) is contacted with an antibody or functional portion thereof which binds to AAH or portion of AAH (e.g., the catalytic domain) under conditions appropriate for binding of the antibody, and antibody binding is monitored. Detection of the antibody, indicative of the formation of a complex between antibody and AAH or a portion thereof, indicates the presence of AAH. Binding of antibody to the cell can be determined as described above under the heading "Binding Assays", for example. The method can be used to detect expression of AAH in cells from an individual (e.g., in a tumor biopsy sample). A quantitative expression of AAH on the surface of tumor cells can be evaluated, for instance, by flow cytometry, and the staining intensity can be correlated with disease progression or risk.
[0123]AAH has a role in cell motility, and so anti-AAH antibodies can be used to inhibit (reduce or prevent) tumor growth or metastasis. Accordingly, the antibodies of the present invention can also be used to modulate AAH function in research and therapeutic applications. For instance, the antibodies described herein can act as inhibitors to inhibit (reduce or prevent) (a) binding (e.g., of an EGF-like domain of a protein) to AAH, (b) a receptor signaling function mediated by AAH, and/or (c) a stimulatory function (e.g., of a substrate of AAH or an AAH-pathway). Antibodies which act as inhibitors of receptor function can block AAH binding directly or indirectly (e.g., by causing a conformational change). Thus, the present invention provides a method of inhibiting cell motility in a mammal (e.g., a human patient), comprising administering to the mammal an effective amount of an antibody or antigen binding portion thereof which binds to a AAH or portion of AAH. Diseases which can be treated according to the method include cancers and can result in amelioration of the disease state. The cancers which can be treated include, but are not limited to solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract (e.g., renal, urothelial cells), liver, pharynx, prostate, ovary, cholangiocarcinomas, as well as adenocarcinomas which include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, neuroectodermal tumors, and so forth. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention.
[0124]The subject method can be useful in treating malignancies of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract, prostate, ovary, pharynx, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. Exemplary solid tumors that can be treated include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lyrnphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
[0125]Patients amenable to treatment with the anti-AAH antibodies described herein may be described as having cancer or a "carcinoma." Carcinomas are recognized by those of ordinary skill in the art as malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is recognized by those skilled in the art and refers to malignant tumors of mesenchymal derivation.
[0126]The methods of the invention can also be used to inhibit the proliferation of hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. For instance, the present invention contemplates the treatment of various mycloid disorders including, but not limited to, acute promyeloid leukemia (APML), acute myclogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, Crit Rev. in Oncol/Hemotol. 11:267-97, 1991). Lymphoid malignancies which may be treated by the subject method include, but are not limited to acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas contemplated by the treatment method of the present invention include, but are not limited to, non-Hodgkin's lymphoma and variants thereof, peripheral T-cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin's disease.
Modes of Administration
[0127]According to the method, one or more antibodies or antigen binding portions thereof can be administered to the host by an appropriate route, either alone or in combination with (before, simultaneous with, or after) another drug. For example, the antibodies of the present invention can also be used in combination with other monoclonal or polyclonal antibodies or with chemotherapeutic treatments.
[0128]An effective amount of an antibody (i.e., one or more antibodies or fragments) is administered. An effective amount is an amount sufficient to achieve the desired therapeutic effect, under the conditions of administration, such as an amount sufficient for inhibition of an AAH function, and thereby, inhibition of a tumor cell.
[0129]A variety of routes of administration are possible including, but not necessarily limited to, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), oral, dietary, topical, inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), depending on the disease or condition to be treated. Other suitable methods of administration can also include rechargeable or biodegradable devices and slow release polymeric devices. The pharmaceutical compositions described herein can also be administered as part of a combinatorial therapy with other agents.
[0130]Formulation of an antibody or portion thereof to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule). An appropriate pharmaceutical composition comprising an antibody or antigen binding portion thereof to be administered can be prepared in a physiologically acceptable vehicle or carrier. A mixture of antibodies and/or portions can also be used. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The antibodies can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired. For inhalation, the compound can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).
Examples
[0131]The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1
Library Screen for Anti-HAAH Antibodies
[0132]A yeast surface display technique (Boder and Wittrup, Nat Biotechnol. 15(6):553-7, 1997) was used to screen a naive human single-chain Fv library for anti-HAAH Fv fragments. This library was prepared as described in Feldhaus et al. (Nature Biotech. 21:163-170, 2003). Briefly, human antibody variable genes were cloned by PCR from commercially available spleen and lymph node poly(A) mRNA pooled from 58 adults. Primers to IgG, IgM, K and X were used for first-strand cDNA synthesis. Separate VH and VL libraries were constructed, then assembled together in single-chain Fv (scFv) format by overlap extension PCR. The scFv library was then suboloned for expression as an Aga2p fusion on the yeast surface. The Aga2p-scFv fusion protein is depicted schematically in FIG. 1. A library of approximately 109 scFv fragments was expressed in yeast.
[0133]The library was screened for binding to a recombinant form of HAAH comprising the extracellular domain as described in Boder and Wittrup (Biotechnol Prog. 14(1):55-62, 1998). Briefly, yeast cells expressing Aga2p-scFv fusions are assayed for binding to fluorescent HAAH by flow cytometry (FACS). Binders are selected by sorting and re-analyzed. Six rounds of screening for binding in 300 nM HAAH allowed the identification of 16 unique clones. FACS analysis of binding of three unique clones to HAAH is depicted in FIG. 2. FACS analysis of binding of 8 unique clones to the catalytic domain of HAAH is depicted in FIG. 3. Of these clones, eight bound to the catalytic domain of HAAH.
Example 2
Binding Activity of Anti-HAAH Antibodies
[0134]The affinity of various scFv clones for HAAH was determined by performing a titration in which cells displaying a given scFv are labeled with varying concentrations of unlabeled HAAH, then varying concentrations of labeled HAAH, and detecting the labeling intensity by FACS. The affinities for two representative anti-HAAH scFv clones are depicted in FIG. 4. Clone 5-2 displayed an affinity for HAAH of approximately 500 nm. Clone 6-12 displayed an affinity for HAAH of approximately 150 nM.
[0135]One antibody clone, clone 27, was isolated which bound to full-length HAAH, but not to the catalytic domain of HAAH. FACS analysis of binding of this scFv clone to the catalytic domain of HAAH and full-length HAAH is depicted in FIG. 5.
Example 3
Binding by Soluble Anti-HAAH scFv Fragments
[0136]The clones that bound to the catalytic domain were expressed in soluble form and analyzed for binding and other biological activities. Soluble scFv fragments of six unique clones were analyzed for binding to the catalytic domain of HAAH (FIG. 6), and full-length HAAH (FIG. 7). ScFv fragments were present at 1-2 μM. Clone 11 showed the highest level of non-specific binding in these assays.
[0137]Next, binding by 3 unique scFv clones to H460 human lung carcinoma cells, which express HAAH, was assessed. ScFv fragments were tested at approximately 1 μM. The results of these experiments are depicted in FIG. 8. Clone 11 scFv also showed the highest level of binding in these assays. Clone 12 also bound H460 cells.
Example 4
Inhibition of Cell Motility by Soluble Anti-HAAH scFv Fragments
[0138]Five unique soluble scFv clones and one mouse IgG, 15C7, were tested for inhibition of H460 cell motility (FIG. 9). Cells were placed in serum-free medium in a filter cup with a pore size of approximately 8 microns. The filter cup was placed into medium containing serum, and cells were incubated to allow migration through the filter. After the incubation, cell number on each side of the filter was determined by staining with crystal violet and counting using a microscope and a hemocytometer. Sixty percent of H460 cells were motile in the absence of antibody. Antibodies were present at 2 μM in the motility assays. 15C7 blocked motility by 100%. Only about 10% of cells were motile in the presence of clone 11 scFv. Clones 10 and 13 also showed modest inhibition of cell motility. Inhibition of cell growth by clone 11 was also assayed. Clone 11 did not demonstrate inhibition of H460 growth at 2 μM (data not shown).
Example 5
Error Prone PCR for Affinity Maturation of Anti-HAAH scFv Fragments
[0139]Next, nucleic acids encoding HAAH-binding scFv were mutagenized and reselected for binding in order to produce "affinity-matured" antibodies in vitro. Mutagenesis was performed by subjecting the scFv-encoding DNA to error prone PCR using nucleotide analogs as described in Zaccolo et al. (J. Mol. Biol., 255:589-603, 1996) and Zaccolo et al. (J. Mol. Biol. 285:775-783, 1999). The initial post-mutagenesis (pre-affinity selection) library had approximately 3×106 members. Selected clones isolated from the library after mutagenesis and which contained full-length open reading frames were sequenced. The mean number of amino acid sequence changes in clones derived from clone 11, relative to the input sequences, was 7±8 (out of 266 amino acids total).
[0140]The mutagenized nucleic acids were re-expressed with yeast surface display and screened for binding using FACS as described above. DNA encoding binders was isolated, re-mutagenized, re-expressed and screened, and so forth for six rounds. Five of the six rounds were performed with sequentially decreasing concentrations of HAAH, starting at 1 μM and ending with 200 mM. One round was performed with detection antibodies in the absence of HAAH to deplete clones that bound to the detection antibody. The mean number of amino acid sequence changes in seven clones derived from clone 11 after this sorting step was 23±16, relative to the input sequences. The number of mutations in germline-encoded framework regions ranged from 8 to 35, as compared to 4 mutations in the original clone (relative to the germline sequence).
[0141]The binding of an improved clone 11 scFv mutant was assayed for binding to the catalytic domain of HAAH (0.5 μM) (FIG. 10). The amino acid sequence of this improved clone is depicted in FIG. 11. Amino acid changes relative to the original clone 11 are shown in bold-faced type and underlined.
[0142]HAAH binding of clone 11 mutants from successive rounds of mutagenesis was assayed by FACS as described above. The level of expression of scFv on yeast cells was analyzed in parallel. The results of these experiments are depicted in FIG. 12. As shown in FIG. 12, the mutants produced after four rounds of screening ("1st round mutants") displayed a higher level of binding to HAAH as compared to original clone 11. One clone, 11m1-2, also displayed a uniformly higher level of binding as compared to original clone 11. Binding of other affinity matured clone 11 mutants is shown in FIG. 13. The dissociation rate constant of the best-improved clones (e.g., 11m1-2) was determined to be 220±60 nM, an improvement of two orders of magnitude over the initial, non-mutagenized scFv from which it was derived.
[0143]Five out of seven affinity matured clones derived from clone 11 had a mutation of cysteine to arginine or tyrosine at position H22 (according to Kabat numbering) in the heavy chain. This cysteine forms an intra-domain disulfide bond. Of the five clones with this mutation, three of them have an addition of a cysteine, with the occurrence of the cysteine residue in either the heavy chain framework region 3 (FW3) or in the light chain CDR3. Of the two clones that did not have a cysteine H22 mutation, one lacked additional cysteines. The other had a replacement of a tyrosine with a cysteine in the heavy chain CDR3 loop, four residues away from cysteine H22. The frequent cysteine mutations may indicate that relaxation of the heavy chain structure can lead to improved binding.
[0144]Affinity maturation was also performed with clone 13. Binding of a clone 13 mutant, 13m1, to the catalytic domain of HAAH is shown in FIG. 14. 13m1 was generated after five cycles of mutagenesis/screening. The amino acid sequence of this mutant is depicted in FIG. 15. Amino acid changes relative to the original clone 13 are bold and underlined. Binding of successive mutants of clone 13 was assayed as described for the clone 11 mutants. As shown in FIG. 16, clone 13m1, and a clone generated from 13m1, 12m2-3, both displayed an enhanced level of binding relative to original clone 13.
Example 6
Chain Shuffling for Affinity Maturation of Anti-HAAH scFv Fragments
[0145]Chain shuffling is a mutagenesis technique whereby an entire antibody chain or portion of a chain is recombined with other chains. In order to generate anti-HAAH scFv fragments with increased affinity for HAAH, a library was constructed by shuffling heavy chains from the naive library against the wild type anti-HAAH light chain (to preserve the light chain sequence and link it with different heavy chain sequences). Specifically, heavy chain fragments from the human naive library were extracted by restriction digestion with NheI and BamHI and ligated into a NheI-BamHI-digested yeast display plasmid containing the light chain of the wild type scFv as depicted in FIG. 17. The initial library generated by this process had approximately 1.2×105 members. Five clones from the initial library were sequenced, and all had heavy chains which were different from the initial heavy chain. FIG. 18A contains a chart listing the heavy chains and light chains in each clone.
[0146]Next, six rounds of FACS screening were performed in the presence of decreasing concentrations of antigen (from 800 nM to 500 nM of HAAH) to screen for improved binders. An additional step of screening in the absence HAAH was performed to eliminate clones binding to the detection antibodies. The scFv display level (X-axis) and HAAH binding (Y-axis) of yeast cells from the pool of mutagenized cells was analyzed by FACS. FIG. 18B shows that clones with high levels of high-affinity scFv fragments were present in the pool.
[0147]Twenty clones were sequenced after the sixth round of screening. Eleven of the clones that were sequenced were unique and each contained a different heavy chain from the wild type scFv. The scFv display level (X-axis) and HAAH binding (Y-axis) of these clones is shown in FIGS. 19A-19K. The dissociation constants for two selected clones, LLm11 and LLm13, were determined to be 240±70 nM and 160±50 nM respectively, which is an improvement of over two orders of magnitude with respect to wild type.
[0148]The framework sequences of the clones generated by this technique had minimal deviation from the germline framework sequences (on average 4±2 mutations from heavy chain germline framework), rendering them less likely to be immunogenic than clones with a greater number of framework mutations. Because all these clones have different heavy chain CDR3, they may have slightly different binding specificities. Multiple binders having different specificities against the same target can be readily screened for secondary desired properties such as minimal binding towards particular human tissues.
Example 7
Affinity Maturation of Anti-HAAH scFv Fragments Using CDR Shuffling
[0149]A third technique, CDR shuffling (also referred to as domain shuffling) was used to generate scFv clones with enhanced affinity for HAAH. To perform CDR shuffling, yeast cells are co-transformed with an acceptor vector and multiple linear insert sequences. The acceptor vector and the insert sequences are designed to have homologous sequences at one or both of their 5' and 3' ends. Homologous recombination between these sites, and at random sites within the gene of interest (e.g., antibody chain) generates "shuffled" products. Further details for performing CDR shuffling are described in Swers et al. (Nucl. Acids Res. 32(3):e36, 2004).
[0150]CDR shuffling was performed with DNA encoding the wild type clone 11 scFv fragment as the acceptor vector, and sequences from a human scFv library as the insert sequences (FIG. 21). The insert sequences had a light chain identical to the clone 11 light chain and the insert sequences of each of three libraries contained regions corresponding to HC FW1 to CDR1, HC FW1 to CDR2, or HC FW1 to CDR3, replaced with naive heavy chain gene sequences from the same VH family as the original clone. The libraries containing replacements of HC FW1 to CDR1 and HC FW1 to CDR2 regions result in a mutagenesis event similar to receptor editing, which naturally occurs in B cells in vivo.
[0151]The libraries with HC FW1 to CDR1, HC FW1 to CDR2, or HC FW1 to CDR3 inserts contained 1.1×104, 1.2×104, and 1.1×104 members, respectively. The libraries were pooled for screening. Six rounds of screening by FACS were performed with decreasing concentrations of antigen, ranging from 500 nM to 160 nM of HAAH. An additional step of screening in the absence HAAH was performed to eliminate clones binding to the detection antibodies.
[0152]Ten clones were isolated and sequenced, and four of the clones were unique. The scFv display level (X-axis) and HAAH binding of these clones (Y-axis) is shown in FIGS. 22A-22D. The amino acid sequences of the CDRs of the wild type clone and the four mutagenized clones are shown in FIG. 23. Of the four unique sequences, three had a replacement in the region from FW1 to CDR3 with naive library sequences. The fourth clone had a replacement of the FW1 to CDR2 sequence with a naive library sequence. The dissociation constant of this mutant was 1 μM-2 μM, an order of magnitude improvement over the original wild type clone. Only two amino acid positions in the framework regions of this mutant differed from the germline framework regions.
[0153]The dissociation constants of various clones were determined. Binding vs. concentration of each clone is shown in FIG. 24. The KD measurements for each clone compared in the graph are as follows:
TABLE-US-00001 Wild type clone 6-22: >10 μM Clone LLm13: 26 ± 8 nM Clone CM4: 17 to 5 nM Clone C4m18: 0.6 ± 0.1 nM
Example 8
Expression of Selected scFv Fragments as IgG Antibodies
[0154]Full length IgG antibodies were created from scFv fragments by inserting the isolated variable heavy and variable light chain sequences into an expression vector containing a sequence encoding the constant regions of the heavy and light IgG chains, the resulting vector expressing a full length IgG antibody (FIG. 25). In brief, the heavy and light chain sequences of the selected scFv fragments were separately amplified by PCR. The resulting fragments were then ligated into the expression vector pPNL501 at the appropriate locations (Pacific Northwest National Laboratory). The resulting vectors were cloned, isolated, and sequenced to confirm the correct sequences were inserted. The vectors were then transfected into COS-7L cells (Invitrogen) by standard methods. These resulting COS-7L cells express and secrete IgG into the culture medium. The medium was then isolated, and the IgG was purified using a Protein A resin by standard methods.
[0155]Binding specificity of the full length IgG antibodies was determined by ELISA and by binding to tumor cells expressing HAAH. For determination of binding specificity by ELISA, either HAAH or bovine serum albumin (BSA) was coated on an ELISA well, and different amounts of IgG were allowed to bind in each well. Detection was performed with a goat anti-human IgG-peroxidase conjugate and a chemoluminescent substrate. The results of this experiment for IgG 6-22, 6-23, 6-25, and 6-28 are shown in FIG. 26. For determination of binding specificity to HAAH-expressing tumor cells, different amounts of IgG antibodies were allowed to bind to H460 tumor cells, which express high levels of HAAH. Binding of IgG antibodies to the cells was detected using a goat anti-human IgG-FITC conjugate with detection by FACS. Results for IgG 6-22 and 6-23 are shown in FIG. 27; results for IgG LLm11, LLm13, LLm15, and CDRm4 are shown in FIG. 28.
[0156]The dissociation constants of IgG 6-22 and CDRm4 were determined. Different concentrations of IgG were allowed to bind either H460 or FOCUS cells at 4° C. FOCUS cells are a hepatocellular carcinoma line which also expresses HAAH. After allowing IgG to bind, the cells were labeled with goat anti-human IgG-phycoerythrin conjugates and fluorescence was detected by FACS. Binding vs. concentration for 6-22 IgG is shown in FIG. 29; binding vs. concentration for CDRm4 IgG is shown in FIG. 30. The KD measurements determined are as follows:
TABLE-US-00002 H460 FOCUS 6-22 IgG 1.3 ± 0.2 nM 1.1 ± 0.2 nM CDRm4 IgG 1.0 ± 0.1 nM 0.7 ± 0.1 nM
[0157]Experiments were performed to determine if the anti-HAAH scFv fragments and derived IgG antibodies bind to similar epitopes on HAAH. FLAG-tagged scFv fragment 6-22 was allowed to bind H460 cells in the presence of 6-22 IgG or a control IgG Binding of the tagged scFv 6-22 was determined by labeling the FLAG tag, present only on the scFv 6-22, with a biotinylated mouse anti-FLAG antibody followed by binding to a streptavidin-phycoerythrin conjugate. IgG 6-22, but not control IgG, competed off the binding of scFv 6-22, indicating that the IgG form of 6-22 binds a similar epitope on HAAH as the scFv 6-22 (FIG. 31).
[0158]To further demonstrate that the scFv fragments and IgG antibodies bind to similar HAAH epitopes, yeast cells displaying different scFv fragments were allowed to bind 15 nM of HAAH in the presence of competing CDRm4 IgG, competing LLm11 IgG, or buffer alone. Binding of HAAH to cells displaying scFv fragments was detected by FACS as described above. The results, shown in FIG. 32, indicate that the IgG antibodies are capable of competing for binding to HAAH, further indicating that the IgG antibodies and scFv fragments bind similar epitopes.
Example 9
Second Generation Affinity Maturation of an Anti-HAAH scFv Fragment by Error-Prone PCR
[0159]Affinity maturation was performed on the CDRm4 mutant scFv to generate the second generation mutants C4m8 and C4m18. As described above, mutagenesis was performed by subjecting the CDRm4 scFv-encoding DNA to error prone PCR using nucleotide analogs. The mutagenized nucleic acids were re-expressed with yeast surface display and screened for binding using FACS as described above. DNA encoding binders was isolated, re-mutagenized, re-expressed and screened, and so forth for four rounds as described above. From this process, the improved second generation scFv clones were isolated.
[0160]The binding of improved clone C4m18 scFv mutant displayed on yeast was assayed for binding to HAAH as described above. This improved mutant was improved approximately 2-fold in both binding and display over CDRm4 (FIG. 33).
[0161]A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Sequence CWU
1
3191125PRTArtificial SequenceSynthetically generated peptide 1Gln Val Gln
Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5
10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser
Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45Trp Leu Gly Arg Thr Tyr Tyr
Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu
Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Tyr Cys Ala Arg Thr Gly Tyr Ser Ser
Ser Trp Val Val Asn Phe 100 105
110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 1252112PRTArtificial SequenceSynthetically
generated peptide 2Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr
Pro Gly Gln1 5 10 15Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20
25 30Tyr Val Tyr Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ala Ala
Ser Leu Ala Ile Ser Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95Arg
Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser Gly
100 105 1103127PRTArtificial
SequenceSynthetically generated peptide 3Gln Val Gln Leu Val Glu Ser Glu
Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Val Ser Tyr Asp Gly Ser Gln Asp Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg
Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90
95Ala Lys Val Gly Arg Ser Ser Asn Trp Phe Ser Arg Tyr
Tyr Tyr Tyr 100 105 110Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 1254109PRTArtificial SequenceSynthetically
generated peptide 4Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser His Tyr 20
25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Val Leu Ile 35 40
45Tyr Asp Val Ala Asn Arg Ala Ala Gly Thr Pro Ala Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Gln 85 90 95Thr
Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Gly 100
1055124PRTArtificial SequenceSynthetically generated peptide 5Gln
Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala
Ile Ser Gly Asp Ser Val Ser Ser Asp 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Lys Ser Arg Ile Ser Ile Asn Pro Asp Thr
Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Ala
Gln Asn Asn Ile Ala Val Ala Gly Phe Asp 100
105 110Tyr Trp Gly Leu Gly Thr Leu Val Thr Val Ser Ser
115 1206110PRTArtificial SequenceSynthetically
generated peptide 6Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr
Pro Gly Gln1 5 10 15Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20
25 30Tyr Val Tyr Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Thr Leu Leu 35 40
45Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg65 70 75
80Ser Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95Ser
Gly Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Ser 100
105 1107123PRTArtificial SequenceSynthetically
generated peptide 7Gln Val Gln Leu Val Glu Ser Glu Gly Gly Val Val Gln
Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Thr Tyr 20
25 30Ala Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Ser Asn Asp Gly Gly His Lys Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asp Ser Met Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr His Cys 85 90 95Ala
Lys Gly Arg Pro Trp Tyr Asp Pro Gly Ala Glu Tyr Phe Gln His
100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 1208111PRTArtificial
SequenceSynthetically generated peptide 8Gln Ser Ala Leu Ile Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln1 5 10
15Trp Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly
Gly Tyr 20 25 30Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45Leu Ile Tyr Asp Val Ser Asp Arg Pro Ser Gly
Val Ser Asn Arg Phe 50 55 60Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65
70 75 80Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90
95Asn Thr Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Ser 100 105
1109128PRTArtificial SequenceSynthetically generated peptide 9Gln Val Gln
Leu Val Gln Ser Glu Gly Gly Val Val Arg Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Gly Asp Tyr 20 25
30Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Ile Asn Trp Asn Gly
Gly Ser Thr Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Val Ser Ser Gly Trp Pro Tyr Tyr
Ser Leu Asp Val Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr
115 120 12510112PRTArtificial
SequenceSynthetically generated peptide 10Gln Ser Ala Leu Ile Gln Pro Arg
Ser Val Ser Gly Ser Pro Gly Gln1 5 10
15Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly
Gly Tyr 20 25 30Asn Tyr Val
Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Gln Leu 35
40 45Met Ile Tyr Asp Val Thr Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65
70 75 80Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Cys Ser Tyr Ala Gly Asp 85 90
95Tyr Thr Tyr Ala Val Phe Gly Thr Gly Thr Gln Leu Thr
Val Leu Ser 100 105
11011126PRTArtificial SequenceSynthetically generated peptide 11Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Gly Tyr 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Ala Ile Ser Phe
Asp Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Arg Asn Thr
Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Lys Val Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95Ala Arg Asp Lys Ser Gly His
Phe Gly Met Asp Val Trp Gly Gln Gly 100 105
110Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 12512107PRTArtificial
SequenceSynthetically generated peptide 12Glu Ile Val Met Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
Pro Asp Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Gly Ser Pro Ile 85 90
95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 10513127PRTArtificial SequenceSynthetically generated
peptide 13Gln Val Gln Leu Val Glu Ser Glu Gly Gly Val Val Gln Pro Gly
Arg1 5 10 15Pro Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20
25 30Ala Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40
45Ala Val Val Ser His Asp Gly Ser Arg Asp Arg Tyr Ala Gly Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr
Tyr Cys 85 90 95Ala Arg
Val Gly Arg Ser Ser Asn Trp Phe Ser Arg Tyr Tyr Tyr Tyr 100
105 110Gly Met Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120
12514107PRTArtificial SequenceSynthetically generated peptide 14Gly Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Pro Pro Arg1 5
10 15Glu Arg Ala Thr Leu Ser Cys Gly
Thr Ser Gln Asn Val Ser His Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Arg Val Leu
Thr 35 40 45Tyr Asp Val Ala Asn
Arg Ala Ala Gly Thr Pro Ala Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Pro Ala Ile Ser Ser Leu
Glu Pro65 70 75 80Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Gln
85 90 95Ala Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys 100 10515127PRTArtificial
SequenceSynthetically generated peptide 15Gln Val Gln Leu Val Glu Ser Glu
Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser
Ser His 20 25 30Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Ser His Asp Gly Ser Arg Asp Arg
Tyr Ala Gly Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg
Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90
95Ala Arg Val Gly Arg Ser Ser Asn Trp Phe Ser Arg Tyr
Tyr Tyr Tyr 100 105 110Asp Met
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 12516113PRTArtificial SequenceSynthetically
generated peptide 16Gly Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu
Pro Pro Arg1 5 10 15Glu
Arg Val Thr Leu Ser Cys Gly Thr Ser Gln Asn Val Ser His His 20
25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Arg Ala Pro Arg Val Leu Thr 35 40
45Tyr Asp Val Ala Asn Lys Ala Ala Gly Thr Pro Ala Arg Phe Ser Gly
50 55 60Ser Gly Ser Glu Thr Asp Phe Thr
Pro Ala Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Gln 85 90 95Ala
Phe Ser Pro Gly Thr Lys Val Asp Ile Lys Ser Gly Ser Glu Gln
100 105 110Lys17127PRTArtificial
SequenceSynthetically generated peptide 17Gln Val Gln Leu Val Glu Ser Glu
Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser His 20 25 30Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Val Ser His Asp Gly Ser Arg Asp Arg
Tyr Ala Gly Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg
Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90
95Ala Arg Val Gly Arg Ser Ser Asn Trp Phe Ser Arg Tyr
His Tyr Tyr 100 105 110Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 12518107PRTArtificial SequenceSynthetically
generated peptide 18Gly Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu
Pro Pro Arg1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Gly Thr Ser Gln Asn Val Ser His Tyr 20
25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Arg Ala Pro Arg Val Leu Thr 35 40
45Tyr Asp Val Ala Asn Arg Ala Ala Gly Thr Pro Ala Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Pro Ala Ile Ser Ser Leu Glu Leu65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Gln 85 90 95Ala
Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100
10519125PRTArtificial SequenceSynthetically generated peptide 19Gln Val
Gln Leu Gln Gln Ser Gly Pro Gly Pro Val Lys Pro Ser Gln1 5
10 15Thr Leu Ser Leu Thr Arg Ala Ile
Pro Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Phe Leu Ser Arg Gly Leu
Glu 35 40 45Trp Leu Gly Arg Thr
Tyr Tyr Arg Ser Lys Arg Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser
Lys Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr Gly
Tyr Ser Ser Ser Trp Val Val Asn Phe 100 105
110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12520111PRTArtificial
SequenceSynthetically generated peptide 20Gln Ser Val Leu Thr Gln Ser Pro
Ser Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn 20 25 30Tyr Val Tyr
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45Ile Tyr Lys Asp Asn Gln Arg Pro Ser Gly Val
Pro Asp Arg Phe Ser 50 55 60Gly Ser
Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly Leu Gln65
70 75 80Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90
95Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val
Leu Ser 100 105
11021123PRTArtificial SequenceSynthetically generated peptide 21Gln Val
Gln Leu Gln Gln Pro Gly Pro Gly Leu Val Glu Pro Ser Gln1 5
10 15Thr Leu Pro Leu Thr Arg Ala Ile
Ser Gly Asp Ser Val Ser Gly Asn 20 25
30Gly Ala Ala Trp Ser Trp Ile Arg Gln Pro Pro Pro Arg Gly Leu
Gly 35 40 45Trp Pro Gly Arg Thr
Tyr Tyr Arg Pro Lys Arg Arg Asn Gly Tyr Ala 50 55
60Val Pro Ala Lys Ser Arg Met Thr Ile Ser Pro Asp Thr Ser
Lys Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr Gly
His Ser Ser Ser Trp Val Val Ser Phe 100 105
110Asp His Trp Gly Gln Gly Ala Thr Ile Ser Ser 115
12022112PRTArtificial SequenceSynthetically generated
peptide 22Ser Gln Pro Val Pro Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro
Gly1 5 10 15Gln Arg Val
Thr Val Ser Cys Ser Gly Ser Ser Ser Asn Ile Glu Ser 20
25 30Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro
Gly Ala Ala Pro Arg Leu 35 40
45Phe Ile His Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Ser 50
55 60Ser Ser Ser Lys Ser Gly Thr Ala Ala
Ser Leu Ala Ile Ser Gly Leu65 70 75
80Gln Ser Lys Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser 85 90 95Leu Arg
Ser Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser 100
105 11023123PRTArtificial
SequenceSynthetically generated peptide 23Gln Val Gln Leu Gln Arg Ser Gly
Pro Gly Leu Val Lys Pro Pro Gln1 5 10
15Ala Pro Ser Leu Thr Cys Ala Val Ser Gly Asp Ser Val Ser
Ser Asn 20 25 30Ser Ala Ala
Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Ile His Tyr Gly Ser Arg Trp
Tyr Asn Asp Tyr Ala 50 55 60Ala Ser
Ala Lys Ser Arg Val Thr Ile Asn Pro Asp Thr Pro Lys Gly65
70 75 80Gln Leu Pro Pro Gln Leu Ser
Pro Val Thr Pro Lys Asp Ala Ala Ala 85 90
95Tyr Tyr Cys Ala Arg Thr Glu Cys Ser Ser Ser Trp Val
Val Asn Phe 100 105 110Gly Tyr
Trp Gly Gln Gly Ala Pro Val Thr Asp 115
12024111PRTArtificial SequenceSynthetically generated peptide 24Gln Pro
Ala Leu Ile Gln Ser Pro Pro Val Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Pro Cys Ser Gly
Ser Ser Phe Asn Ile Gly Ser Asn 20 25
30His Val Tyr Trp His Gln Gln Pro Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Val His Lys Ser Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Ser Pro 50 55
60Gly Pro Arg Pro Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Leu Gly Ala
Gly Thr Lys Leu Thr Ala Arg Ser 100 105
11025125PRTArtificial SequenceSynthetically generated peptide
25Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Pro Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr His Arg Phe Lys Trp Tyr Asn Asp His Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr
Gly Tyr Ser Ser Ser Trp Val Val Asn Phe 100
105 110Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser
Ser 115 120 12526111PRTArtificial
SequenceSynthetically generated peptide 26Gln Pro Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn 20 25 30Tyr Val Tyr
Arg Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35
40 45Ile Tyr Lys Asn Asn Gln Arg Ser Ser Gly Val
Pro Ala Arg Phe Ser 50 55 60Gly Pro
Lys Ser Gly Ala Ala Ala Pro Leu Thr Thr Ser Gly Leu Gln65
70 75 80Ser Gly Asp Glu Ala Gly Tyr
Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90
95Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val
Leu Ser 100 105
11027125PRTArtificial SequenceSynthetically generated peptide 27Gln Val
Gln Leu Gln Trp Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5
10 15Thr Leu Ser Leu Thr Arg Ala Ile
Ser Gly Asn Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Ser Trp Ile Arg Gln Ser Leu Ser Arg Gly Leu
Glu 35 40 45Trp Leu Gly Arg Thr
Tyr Tyr Gly Ser Lys Arg Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Pro
Lys Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Ala Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr Gly
Tyr Ser Gly Ser Arg Val Val Asn Phe 100 105
110Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12528109PRTArtificial
SequenceSynthetically generated peptide 28Gln Pro Val Leu Thr Gln Ser Pro
Pro Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn 20 25 30Tyr Val His
Arg Tyr Gln Gln Pro Pro Gly Ala Ala Pro Glu Leu Leu 35
40 45Ile His Lys Asn Asn Gln Arg Pro Ser Gly Val
Pro Asp Arg Phe Ser 50 55 60Gly Ser
Lys Ser Gly Thr Ala Asp Ala Ile Ser Gly Pro Gln Ser Glu65
70 75 80Asp Glu Ala Asn Tyr Tyr Cys
Val Ala Trp Asp Asp Ser Pro Cys Gly 85 90
95Tyr Val Phe Gly Ala Gly Thr Lys Leu Thr Val Leu Ser
100 10529125PRTArtificial SequenceSynthetically
generated peptide 29Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln1 5 10 15Thr
Phe Ser Leu Thr Tyr Ala Val Ser Gly Gly Ser Ala Ser Ser Asn 20
25 30Ser Ala Ala Trp Asn Trp Ile Arg
Gln Ser Leu Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60Val Ser Val Lys Ser Arg Thr Thr
Ile Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Gly Asp
Ala Ala Val 85 90 95Cys
Tyr Cys Ala Gly Ala Gly Tyr Ser Ser Ser Arg Ala Val Asn Phe
100 105 110Asp Cys Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120
12530111PRTArtificial SequenceSynthetically generated peptide 30Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Lys Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80Pro
Glu Gly Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Leu Gly Thr
Gly Thr Lys Leu Thr Val Leu Ser 100 105
11031264PRTArtificial SequenceSynthetically generated peptide
31Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr
Gly Tyr Ser Ser Ser Trp Val Val Asn Phe 100
105 110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Ser Ala 115 120 125Ser Ala
Pro Thr Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly 130
135 140Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val
Leu Thr Gln Ser Pro145 150 155
160Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly
165 170 175Ser Ser Ser Asn
Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu 180
185 190Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys
Asn Asn Gln Arg Pro 195 200 205Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ala Ala 210
215 220Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu
Asp Glu Ala Asp Tyr Tyr225 230 235
240Cys Ala Ala Trp Asp Asp Ser Leu Arg Gly Tyr Val Phe Gly Thr
Gly 245 250 255Thr Lys Leu
Thr Val Leu Ser Gly 26032256PRTArtificial
SequenceSynthetically generated peptide 32Gln Val Gln Leu Val Glu Ser Glu
Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Val Ser Tyr Asp Gly Ser Gln Asp Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg
Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90
95Ala Lys Val Gly Arg Ser Ser Asn Trp Phe Ser Arg Tyr
Tyr Tyr Tyr 100 105 110Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 115
120 125Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly
Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu145
150 155 160Ser Pro Gly Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val 165
170 175Ser His Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg 180 185 190Val
Leu Ile Tyr Asp Val Ala Asn Arg Ala Ala Gly Thr Pro Ala Arg 195
200 205Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser 210 215
220Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn225
230 235 240Trp Pro Gln Thr
Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Gly 245
250 25533257PRTArtificial SequenceSynthetically
generated peptide 33Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asp 20
25 30Ser Ala Ala Trp Asn Trp Ile Arg
Gln Ser Pro Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60Val Ser Val Lys Ser Arg Ile Ser
Ile Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp
Thr Ala Val 85 90 95Tyr
Tyr Cys Ala Arg Ala Gln Asn Asn Ile Ala Val Ala Gly Phe Asp
100 105 110Tyr Trp Gly Leu Gly Thr Leu
Val Thr Val Ser Ser Gly Ile Leu Gly 115 120
125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 130 135 140Gln Pro Val Leu Thr Gln
Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln145 150
155 160Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser
Asn Ile Gly Ser Asn 165 170
175Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Thr Leu Leu
180 185 190Ile Tyr Arg Asn Asn Gln
Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 195 200
205Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu Arg 210 215 220Ser Glu Asp Glu Ala
Glu Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu225 230
235 240Ser Gly Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu Ser Gly Ile 245 250
255Leu34257PRTArtificial SequenceSynthetically generated peptide
34Gln Val Gln Leu Val Glu Ser Glu Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Gly Thr Tyr 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Val Ile
Ser Asn Asp Gly Gly His Lys Tyr Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asp Ser Met Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr His Cys
85 90 95Ala Lys Gly Arg Pro Trp
Tyr Asp Pro Gly Ala Glu Tyr Phe Gln His 100
105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly
Ile Leu Gly Ser 115 120 125Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130
135 140Ser Ala Leu Ile Gln Pro Ala Ser Val Ser Gly
Ser Pro Gly Gln Trp145 150 155
160Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn
165 170 175Tyr Val Ser Trp
Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Leu 180
185 190Ile Tyr Asp Val Ser Asp Arg Pro Ser Gly Val
Ser Asn Arg Phe Ser 195 200 205Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln 210
215 220Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser
Ser Tyr Thr Ser Ser Asn 225 230
235 240Thr Val Leu Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Ser Gly Ile 245 250
255Leu35263PRTArtificial SequenceSynthetically generated peptide
35Gln Val Gln Leu Val Gln Ser Glu Gly Gly Val Val Arg Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25
30Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Ser Ile
Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95Ala Arg Val Ser Ser Gly
Trp Pro Tyr Tyr Ser Leu Asp Val Trp Gly 100
105 110Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Ala
Ser Ala Pro Thr 115 120 125Gly Ile
Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130
135 140Gly Gly Gly Ser Gln Ser Ala Leu Ile Gln Pro
Arg Ser Val Ser Gly145 150 155
160Ser Pro Gly Gln Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
165 170 175Val Gly Gly Tyr
Asn Tyr Val Ser Trp Tyr Gln His His Pro Gly Lys 180
185 190Ala Pro Gln Leu Met Ile Tyr Asp Val Thr Lys
Arg Pro Ser Gly Val 195 200 205Pro
Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr 210
215 220Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Cys Ser225 230 235
240Tyr Ala Gly Asp Tyr Thr Tyr Ala Val Phe Gly Thr Gly Thr Gln
Leu 245 250 255Thr Val Leu
Ser Gly Ile Leu 26036257PRTArtificial SequenceSynthetically
generated peptide 36Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20
25 30Ala Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Ala Ile Ser Phe Asp Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Val Ser Arg
Asp Asn Ser Arg Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Lys Val Asp Asp Thr Ala Val
Tyr Phe Cys 85 90 95Ala
Arg Asp Lys Ser Gly His Phe Gly Met Asp Val Trp Gly Gln Gly
100 105 110Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Gly Ile 115 120
125Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly 130 135 140Gly Ser Glu Ile Val Met
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser145 150
155 160Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser 165 170
175Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
180 185 190Leu Leu Ile Tyr Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg 195 200
205Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg 210 215 220Leu Glu Pro Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser225 230
235 240Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu
Glu Ile Lys Ser Gly Ile 245 250
255Leu37256PRTArtificial SequenceSynthetically generated peptide
37Gln Val Gln Leu Val Glu Ser Glu Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Pro Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Val Val
Ser His Asp Gly Ser Arg Asp Arg Tyr Ala Gly Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Tyr Cys
85 90 95Ala Arg Val Gly Arg Ser
Ser Asn Trp Phe Ser Arg Tyr Tyr Tyr Tyr 100
105 110Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser Gly 115 120 125Ile Leu
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Asp Ser Gly Gly 130
135 140Gly Gly Ser Gly Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu145 150 155
160Pro Pro Arg Glu Arg Ala Thr Leu Ser Cys Gly Thr Ser Gln Asn Val
165 170 175Ser His Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Arg 180
185 190Val Leu Thr Tyr Asp Val Ala Asn Arg Ala Ala
Gly Thr Pro Ala Arg 195 200 205Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Pro Ala Ile Ser Ser 210
215 220Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Arg Ser Asn225 230 235
240Trp Pro Gln Ala Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser
Gly 245 250
25538264PRTArtificial SequenceSynthetically generated peptide 38Gln Val
Gln Leu Val Glu Ser Glu Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Pro Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Ala Phe Ser Ser His 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Ser His
Asp Gly Ser Arg Asp Arg Tyr Ala Gly Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Tyr Cys
85 90 95Ala Arg Val Gly Arg Ser Ser
Asn Trp Phe Ser Arg Tyr Tyr Tyr Tyr 100 105
110Asp Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Gly 115 120 125Ile Leu Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Asp Ser Gly Gly 130
135 140Gly Gly Ser Gly Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu145 150 155
160Pro Pro Arg Glu Arg Val Thr Leu Ser Cys Gly Thr Ser Gln Asn Val
165 170 175Ser His His Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Arg 180
185 190Val Leu Thr Tyr Asp Val Ala Asn Lys Ala Ala Gly
Thr Pro Ala Arg 195 200 205Phe Ser
Gly Ser Gly Ser Glu Thr Asp Phe Thr Pro Ala Ile Ser Ser 210
215 220Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn225 230 235
240Trp Pro Gln Ala Phe Ser Pro Gly Thr Lys Val Asp Ile Lys Ser Gly
245 250 255Ser Glu Gln Lys
Leu Ile Ser Glu 26039267PRTArtificial SequenceSynthetically
generated peptide 39Gln Val Gln Leu Val Glu Ser Glu Gly Gly Val Val Gln
Pro Gly Arg1 5 10 15Pro
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20
25 30Ala Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Val Ser His Asp Gly Ser Arg Asp Arg Tyr Ala Gly Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Val
Tyr Tyr Cys 85 90 95Ala
Arg Val Gly Arg Ser Ser Asn Trp Phe Ser Arg Tyr His Tyr Tyr
100 105 110Gly Met Asp Val Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Gly 115 120
125Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Asp Ser Gly
Gly 130 135 140Gly Gly Ser Gly Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu145 150
155 160Pro Pro Arg Glu Arg Ala Thr Leu Ser Cys Gly
Thr Ser Gln Asn Val 165 170
175Ser His Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Arg
180 185 190Val Leu Thr Tyr Asp Val
Ala Asn Arg Ala Ala Gly Thr Pro Ala Arg 195 200
205Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Pro Ala Ile
Ser Ser 210 215 220Leu Glu Leu Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn225 230
235 240Trp Pro Gln Ala Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys Ser Gly 245 250
255Ser Glu Gln Lys Leu Ile Ser Glu Glu Ala Leu 260
26540266PRTArtificial SequenceSynthetically generated peptide
40Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Pro Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Arg
Ala Ile Pro Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Phe Leu Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Arg Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr
Gly Tyr Ser Ser Ser Trp Val Val Asn Phe 100
105 110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Ser Ala 115 120 125Ser Ala
Pro Thr Gly Ile Leu Gly Phe Gly Gly Gly Gly Ser Gly Gly 130
135 140Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val
Leu Thr Gln Ser Pro145 150 155
160Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly
165 170 175Ser Ser Ser Asn
Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu 180
185 190Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys
Asp Asn Gln Arg Pro 195 200 205Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ala Ala 210
215 220Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu
Asp Glu Ala Asp Tyr Tyr225 230 235
240Cys Ala Ala Trp Asp Asp Ser Leu Arg Gly Tyr Val Phe Gly Thr
Gly 245 250 255Thr Lys Leu
Thr Val Leu Ser Gly Ile Leu 260
26541264PRTArtificial SequenceSynthetically generated peptide 41Gln Val
Gln Leu Gln Gln Pro Gly Pro Gly Leu Val Glu Pro Ser Gln1 5
10 15Thr Leu Pro Leu Thr Arg Ala Ile
Ser Gly Asp Ser Val Ser Gly Asn 20 25
30Gly Ala Ala Trp Ser Trp Ile Arg Gln Pro Pro Pro Arg Gly Leu
Gly 35 40 45Trp Pro Gly Arg Thr
Tyr Tyr Arg Pro Lys Arg Arg Asn Gly Tyr Ala 50 55
60Val Pro Ala Lys Ser Arg Met Thr Ile Ser Pro Asp Thr Ser
Lys Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr Gly
His Ser Ser Ser Trp Val Val Ser Phe 100 105
110Asp His Trp Gly Gln Gly Ala Thr Ile Ser Ser Gly Asn Ala
Ser Ala 115 120 125Pro Thr Gly Val
Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130
135 140Ser Gly Gly Gly Ser Ser Gln Pro Val Pro Thr Gln
Ser Pro Ser Ala145 150 155
160Ser Gly Thr Pro Gly Gln Arg Val Thr Val Ser Cys Ser Gly Ser Ser
165 170 175Ser Asn Ile Glu Ser
Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly 180
185 190Ala Ala Pro Arg Leu Phe Ile His Lys Asn Asn Gln
Arg Pro Ser Gly 195 200 205Val Pro
Asp Arg Ser Ser Ser Ser Lys Ser Gly Thr Ala Ala Ser Leu 210
215 220Ala Ile Ser Gly Leu Gln Ser Lys Asp Glu Ala
Asp Tyr Tyr Cys Ala225 230 235
240Ala Trp Asp Asp Ser Leu Arg Ser Tyr Val Phe Gly Thr Gly Thr Lys
245 250 255Leu Thr Val Leu
Ser Gly Ile Leu 26042264PRTArtificial SequenceSynthetically
generated peptide 42Gln Val Gln Leu Gln Arg Ser Gly Pro Gly Leu Val Lys
Pro Pro Gln1 5 10 15Ala
Pro Ser Leu Thr Cys Ala Val Ser Gly Asp Ser Val Ser Ser Asn 20
25 30Ser Ala Ala Trp Asn Trp Ile Arg
Gln Ser Leu Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Ile His Tyr Gly Ser Arg Trp Tyr Asn Asp Tyr Ala
50 55 60Ala Ser Ala Lys Ser Arg Val Thr
Ile Asn Pro Asp Thr Pro Lys Gly65 70 75
80Gln Leu Pro Pro Gln Leu Ser Pro Val Thr Pro Lys Asp
Ala Ala Ala 85 90 95Tyr
Tyr Cys Ala Arg Thr Glu Cys Ser Ser Ser Trp Val Val Asn Phe
100 105 110Gly Tyr Trp Gly Gln Gly Ala
Pro Val Thr Asp Gly Ser Ala Pro Ala 115 120
125Pro Thr Gly Ile Leu Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly
Gly 130 135 140Ser Gly Gly Gly Gly Pro
Gln Pro Ala Leu Ile Gln Ser Pro Pro Val145 150
155 160Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Pro
Cys Ser Gly Ser Ser 165 170
175Phe Asn Ile Gly Ser Asn His Val Tyr Trp His Gln Gln Pro Pro Gly
180 185 190Thr Ala Pro Lys Leu Leu
Val His Lys Ser Asn Gln Arg Pro Ser Gly 195 200
205Val Pro Asp Arg Ser Pro Gly Pro Arg Pro Gly Thr Ala Ala
Ser Leu 210 215 220Ala Ile Ser Gly Leu
Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala225 230
235 240Ala Trp Asp Asp Ser Leu Arg Gly Tyr Val
Leu Gly Ala Gly Thr Lys 245 250
255Leu Thr Ala Arg Ser Gly Ile Leu 26043266PRTArtificial
SequenceSynthetically generated peptide 43Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Pro Gly Asp Ser Val Ser
Ser Asn 20 25 30Ser Ala Ala
Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr His Arg Phe Lys Trp
Tyr Asn Asp His Ala 50 55 60Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Asn
Ser Val Thr Pro Glu Asp Thr Ala Val 85 90
95Tyr Tyr Cys Ala Arg Thr Gly Tyr Ser Ser Ser Trp Val
Val Asn Phe 100 105 110Asp Tyr
Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser Gly Ser Ala 115
120 125Ser Ala Pro Thr Gly Val Leu Gly Pro Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly
Gly Ser Gly Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Pro Pro145
150 155 160Ser Ala Ser Gly Thr Pro
Gly Gln Arg Val Thr Ile Ser Cys Ser Gly 165
170 175Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr Arg
Tyr Gln Gln Leu 180 185 190Pro
Gly Ala Ala Pro Lys Leu Leu Ile Tyr Lys Asn Asn Gln Arg Ser 195
200 205Ser Gly Val Pro Ala Arg Phe Ser Gly
Pro Lys Ser Gly Ala Ala Ala 210 215
220Pro Leu Thr Thr Ser Gly Leu Gln Ser Gly Asp Glu Ala Gly Tyr Tyr225
230 235 240Cys Ala Ala Trp
Asp Asp Ser Leu Arg Gly Tyr Val Phe Gly Thr Gly 245
250 255Thr Lys Leu Thr Val Leu Ser Gly Ile Leu
260 26544264PRTArtificial SequenceSynthetically
generated peptide 44Gln Val Gln Leu Gln Trp Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln1 5 10 15Thr
Leu Ser Leu Thr Arg Ala Ile Ser Gly Asn Ser Val Ser Ser Asn 20
25 30Ser Ala Ala Trp Ser Trp Ile Arg
Gln Ser Leu Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Gly Ser Lys Arg Tyr Asn Asp Tyr Ala
50 55 60Val Ser Val Lys Ser Arg Ile Thr
Ile Asn Pro Asp Thr Pro Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp
Ala Ala Val 85 90 95Tyr
Tyr Cys Ala Arg Thr Gly Tyr Ser Gly Ser Arg Val Val Asn Phe
100 105 110Gly Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Gly Arg Ala 115 120
125Pro Ala Pro Thr Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly
Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Pro Gln Pro Val Leu Thr Gln Ser Pro145 150
155 160Pro Ala Ser Gly Thr Pro Gly Gln Arg Val Thr
Ile Ser Cys Ser Gly 165 170
175Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val His Arg Tyr Gln Gln Pro
180 185 190Pro Gly Ala Ala Pro Glu
Leu Leu Ile His Lys Asn Asn Gln Arg Pro 195 200
205Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr
Ala Asp 210 215 220Ala Ile Ser Gly Pro
Gln Ser Glu Asp Glu Ala Asn Tyr Tyr Cys Val225 230
235 240Ala Trp Asp Asp Ser Pro Cys Gly Tyr Val
Phe Gly Ala Gly Thr Lys 245 250
255Leu Thr Val Leu Ser Gly Ile Leu 26045265PRTArtificial
SequenceSynthetically generated peptide 45Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Phe Ser Leu Thr Tyr Ala Val Ser Gly Gly Ser Ala Ser
Ser Asn 20 25 30Ser Ala Ala
Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp
Tyr Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Thr Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Asn
Ser Val Thr Pro Gly Asp Ala Ala Val 85 90
95Cys Tyr Cys Ala Gly Ala Gly Tyr Ser Ser Ser Arg Ala
Val Asn Phe 100 105 110Asp Cys
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ala 115
120 125Ser Thr Pro Thr Gly Ile Leu Gly Ser Gly
Gly Gly Ser Gly Gly Gly 130 135 140Gly
Pro Gly Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser145
150 155 160Ala Ser Gly Thr Pro Gly
Gln Arg Val Thr Ile Ser Cys Ser Gly Ser 165
170 175Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr
Gln Gln Leu Pro 180 185 190Gly
Thr Ala Pro Lys Leu Leu Ile Tyr Lys Asn Asn Gln Arg Pro Ser 195
200 205Gly Val Pro Asp Arg Phe Ser Gly Ser
Lys Ser Gly Thr Ala Ala Ser 210 215
220Leu Ala Ile Ser Gly Leu Gln Pro Glu Gly Glu Ala Asp Tyr Tyr Cys225
230 235 240Ala Ala Trp Asp
Asp Ser Leu Arg Gly Tyr Val Leu Gly Thr Gly Thr 245
250 255Lys Leu Thr Val Leu Ser Gly Ile Leu
260 265467PRTArtificial SequenceSynthetically
generated peptide 46Ser Asn Ser Ala Ala Trp Asn1
54718PRTArtificial SequenceSynthetically generated peptide 47Arg Thr Tyr
Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5
10 15Lys Ser4813PRTArtificial
SequenceSynthetically generated peptide 48Thr Gly Tyr Ser Ser Ser Trp Val
Val Asn Phe Asp Tyr1 5
104913PRTArtificial SequenceSynthetically generated peptide 49Ser Gly Ser
Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr1 5
105012PRTArtificial SequenceSynthetically generated peptide 50Lys Leu
Leu Ile Tyr Lys Asn Asn Gln Arg Pro Ser1 5
105111PRTArtificial SequenceSynthetically generated peptide 51Ala Ala
Trp Asp Asp Ser Leu Arg Gly Tyr Val1 5
10525PRTArtificial SequenceSynthetically generated peptide 52Ser Tyr Ala
Met His1 55317PRTArtificial SequenceSynthetically generated
peptide 53Val Val Ser Tyr Asp Gly Ser Gln Asp Tyr Tyr Ala Asp Ser Val
Lys1 5 10
15Gly5418PRTArtificial SequenceSynthetically generated peptide 54Val Gly
Arg Ser Ser Asn Trp Phe Ser Arg Tyr Tyr Tyr Tyr Gly Met1 5
10 15Asp Val5511PRTArtificial
SequenceSynthetically generated peptide 55Arg Ala Ser Gln Ser Val Ser His
Tyr Leu Ala1 5 10567PRTArtificial
SequenceSynthetically generated peptide 56Asp Val Ala Asn Arg Ala Ala1
5579PRTArtificial SequenceSynthetically generated peptide
57Gln Gln Arg Ser Asn Trp Pro Gln Thr1 5587PRTArtificial
SequenceSynthetically generated peptide 58Ser Asp Ser Ala Ala Trp Asn1
559375PRTArtificial SequenceSynthetically generated peptide
59Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Pro Cys
Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45Ile Tyr Lys
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile
Ser Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly
Thr Gly Thr Lys Leu Thr Val Leu Ser Gly 100
105 110Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly 115 120 125Gly Gly
Ser Arg Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr 130
135 140Pro Gly Gln Arg Val Thr Ile Pro Cys Ser Gly
Ser Ser Ser Asn Ile145 150 155
160Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
165 170 175Lys Leu Leu Ile
Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 180
185 190Arg Phe Ser Gly Ser Lys Ser Gly Thr Ala Ala
Ser Leu Ala Ile Ser 195 200 205Gly
Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp 210
215 220Asp Ser Leu Arg Gly Tyr Val Phe Gly Thr
Gly Thr Lys Leu Thr Val225 230 235
240Leu Ser Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly 245 250 255Ser Gly Gly
Gly Gly Ser Arg Pro Val Leu Thr Gln Ser Pro Ser Ala 260
265 270Ser Gly Thr Pro Gly Gln Arg Val Thr Ile
Pro Cys Ser Gly Ser Ser 275 280
285Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly 290
295 300Thr Ala Pro Lys Leu Leu Ile Tyr
Lys Asn Asn Gln Arg Pro Ser Gly305 310
315 320Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr
Ala Ala Ser Leu 325 330
335Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
340 345 350Ala Trp Asp Asp Ser Leu
Arg Gly Tyr Val Phe Gly Thr Gly Thr Arg 355 360
365Leu Thr Val Leu Ser Gly Ser 370
3756012PRTArtificial SequenceSynthetically generated peptide 60Ala Gln
Asn Asn Ile Ala Val Ala Gly Phe Asp Tyr1 5
1061256PRTArtificial SequenceSynthetically generated peptide 61Gln Val
Gln Leu Val Glu Ser Glu Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Pro Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser His 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Val Ser His
Asp Gly Ser Arg Asp Arg Tyr Ala Gly Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Tyr Cys
85 90 95Ala Arg Val Gly Arg Ser Ser
Asn Trp Phe Ser Arg Tyr Tyr Tyr Tyr 100 105
110Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Gly 115 120 125Ile Leu Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Asp Ser Gly Gly 130
135 140Gly Gly Ser Gly Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu145 150 155
160Pro Pro Arg Glu Arg Ala Thr Leu Ser Cys Gly Thr Ser Gln Asn Val
165 170 175Ser His Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Arg 180
185 190Val Leu Thr Tyr Asp Val Ala Asn Arg Ala Ala Gly
Thr Pro Ala Arg 195 200 205Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Pro Ala Ile Ser Ser 210
215 220Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn225 230 235
240Trp Pro Gln Ala Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Gly
245 250 2556212PRTArtificial
SequenceSynthetically generated peptide 62Thr Leu Leu Ile Tyr Arg Asn Asn
Gln Arg Pro Ser1 5 106310PRTArtificial
SequenceSynthetically generated peptide 63Ala Ala Trp Asp Asp Ser Leu Ser
Gly Val1 5 10645PRTArtificial
SequenceSynthetically generated peptide 64Thr Tyr Ala Met His1
56517PRTArtificial SequenceSynthetically generated peptide 65Val Ile
Ser Asn Asp Gly Gly His Lys Tyr Tyr Ala Asp Ser Val Lys1 5
10 15Gly6614PRTArtificial
SequenceSynthetically generated peptide 66Gly Arg Pro Trp Tyr Asp Pro Gly
Ala Glu Tyr Phe Gln His1 5
106714PRTArtificial SequenceSynthetically generated peptide 67Thr Gly Thr
Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser1 5
10687PRTArtificial SequenceSynthetically generated peptide 68Asp Val
Ser Asp Arg Pro Ser1 56910PRTArtificial
SequenceSynthetically generated peptide 69Ser Ser Tyr Thr Ser Ser Asn Thr
Val Leu1 5 10705PRTArtificial
SequenceSynthetically generated peptide 70Asp Tyr Ala Met Gly1
57117PRTArtificial SequenceSynthetically generated peptide 71Ser Ile
Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val Lys1 5
10 15Gly7212PRTArtificial
SequenceSynthetically generated peptide 72Val Ser Ser Gly Trp Pro Tyr Tyr
Ser Leu Asp Val1 5 107314PRTArtificial
SequenceSynthetically generated peptide 73Thr Gly Thr Ser Ser Asp Val Gly
Gly Tyr Asn Tyr Val Ser1 5
10747PRTArtificial SequenceSynthetically generated peptide 74Asp Val Thr
Lys Arg Pro Ser1 57511PRTArtificial SequenceSynthetically
generated peptide 75Cys Ser Tyr Ala Gly Asp Tyr Thr Tyr Ala Val1
5 10765PRTArtificial SequenceSynthetically
generated peptide 76Gly Tyr Ala Met His1 57717PRTArtificial
SequenceSynthetically generated peptide 77Ala Ile Ser Phe Asp Gly Asn Tyr
Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly7810PRTArtificial SequenceSynthetically generated
peptide 78Asp Lys Ser Gly His Phe Gly Met Asp Val1 5
107912PRTArtificial SequenceSynthetically generated peptide
79Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala1 5
10807PRTArtificial SequenceSynthetically generated peptide
80Gly Ala Ser Ser Arg Ala Thr1 5818PRTArtificial
SequenceSynthetically generated peptide 81Gln Gln Tyr Gly Ser Pro Ile
Thr1 5825PRTArtificial SequenceSynthetically generated
peptide 82Ser His Ala Met His1 58317PRTArtificial
SequenceSynthetically generated peptide 83Val Val Ser His Asp Gly Ser Arg
Asp Arg Tyr Ala Gly Ser Val Lys1 5 10
15Gly8412PRTArtificial SequenceSynthetically generated
peptide 84Cys Gly Thr Ser Gln Asn Val Ser His Tyr Leu Ala1
5 10859PRTArtificial SequenceSynthetically generated
peptide 85Gln Gln Arg Ser Asn Trp Pro Gln Ala1
58617PRTArtificial SequenceSynthetically generated peptide 86Val Ile Ser
His Asp Gly Ser Arg Asp Arg Tyr Ala Gly Ser Val Lys1 5
10 15Gly8718PRTArtificial
SequenceSynthetically generated peptide 87Val Gly Arg Ser Ser Asn Trp Phe
Ser Arg Tyr Tyr Tyr Tyr Asp Met1 5 10
15Asp Val8811PRTArtificial SequenceSynthetically generated
peptide 88Gly Thr Ser Gln Asn Val Ser His His Leu Ala1 5
10897PRTArtificial SequenceSynthetically generated peptide
89Asp Val Ala Asn Lys Ala Ala1 5909PRTArtificial
SequenceSynthetically generated peptide 90Gln Gln Arg Ser Asn Trp Pro Gln
Ala1 59118PRTArtificial SequenceSynthetically generated
peptide 91Val Gly Arg Ser Ser Asn Trp Phe Ser Arg Tyr His Tyr Tyr Gly
Met1 5 10 15Asp
Val9211PRTArtificial SequenceSynthetically generated peptide 92Gly Thr
Ser Gln Asn Val Ser His Tyr Leu Ala1 5
10939PRTArtificial SequenceSynthetically generated peptide 93Gln Gln Arg
Ser Asn Trp Pro Gln Ala1 5948PRTArtificial
SequenceSynthetically generated peptide 94Ser Ser Asn Ser Ala Ala Trp
Asn1 59518PRTArtificial SequenceSynthetically generated
peptide 95Arg Thr Tyr Tyr Arg Ser Lys Arg Tyr Asn Asp Tyr Ala Val Ser
Val1 5 10 15Lys
Ser9613PRTArtificial SequenceSynthetically generated peptide 96Thr Gly
Tyr Ser Ser Ser Trp Val Val Asn Phe Asp Tyr1 5
109719PRTArtificial SequenceSynthetically generated peptide 97Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1
5 10 15Lys Ser Arg9812PRTArtificial
SequenceSynthetically generated peptide 98Lys Leu Leu Ile Tyr Lys Asp Asn
Gln Arg Pro Ser1 5 10997PRTArtificial
SequenceSynthetically generated peptide 99Gly Asn Gly Ala Ala Trp Ser1
510018PRTArtificial SequenceSynthetically generated peptide
100Arg Thr Tyr Tyr Arg Pro Lys Arg Arg Asn Gly Tyr Ala Val Pro Ala1
5 10 15Lys
Ser10113PRTArtificial SequenceSynthetically generated peptide 101Thr Gly
His Ser Ser Ser Trp Val Val Ser Phe Asp His1 5
1010213PRTArtificial SequenceSynthetically generated peptide 102Ser
Gly Ser Ser Ser Asn Ile Glu Ser Asn Tyr Val Tyr1 5
1010312PRTArtificial SequenceSynthetically generated peptide
103Arg Leu Phe Ile His Lys Asn Asn Gln Arg Pro Ser1 5
1010411PRTArtificial SequenceSynthetically generated peptide
104Ala Ala Trp Asp Asp Ser Leu Arg Ser Tyr Val1 5
1010518PRTArtificial SequenceSynthetically generated peptide
105Arg Ile His Tyr Gly Ser Arg Trp Tyr Asn Asp Tyr Ala Ala Ser Ala1
5 10 15Lys
Ser10613PRTArtificial SequenceSynthetically generated peptide 106Thr Glu
Cys Ser Ser Ser Trp Val Val Asn Phe Gly Tyr1 5
1010713PRTArtificial SequenceSynthetically generated peptide 107Ser
Gly Ser Ser Phe Asn Ile Gly Ser Asn His Val Tyr1 5
1010812PRTArtificial SequenceSynthetically generated peptide
108Lys Leu Leu Val His Lys Ser Asn Gln Arg Pro Ser1 5
1010918PRTArtificial SequenceSynthetically generated peptide
109Arg Thr Tyr His Arg Phe Lys Trp Tyr Asn Asp His Ala Val Ser Val1
5 10 15Lys
Ser11012PRTArtificial SequenceSynthetically generated peptide 110Lys Leu
Leu Ile Tyr Lys Asn Asn Gln Arg Ser Ser1 5
101117PRTArtificial SequenceSynthetically generated peptide 111Ser Asn
Ser Ala Ala Trp Ser1 511218PRTArtificial
SequenceSynthetically generated peptide 112Arg Thr Tyr Tyr Gly Ser Lys
Arg Tyr Asn Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser11313PRTArtificial SequenceSynthetically
generated peptide 113Thr Gly Tyr Ser Gly Ser Arg Val Val Asn Phe Gly Tyr1
5 1011413PRTArtificial
SequenceSynthetically generated peptide 114Ser Gly Ser Ser Ser Asn Ile
Gly Ser Asn Tyr Val His1 5
1011512PRTArtificial SequenceSynthetically generated peptide 115Glu Leu
Leu Ile His Lys Asn Asn Gln Arg Pro Ser1 5
1011611PRTArtificial SequenceSynthetically generated peptide 116Val Ala
Trp Asp Asp Ser Pro Cys Gly Tyr Val1 5
1011713PRTArtificial SequenceSynthetically generated peptide 117Ala Gly
Tyr Ser Ser Ser Arg Ala Val Asn Phe Asp Cys1 5
1011813PRTArtificial SequenceSynthetically generated peptide 118Thr
Gly Tyr Ser Ser Ser Trp Val Val Asn Phe Asp Tyr1 5
10119131PRTArtificial SequenceSynthetically generated peptide
119Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Ile 20 25
30Thr Ala Ala Trp Asn Trp Leu Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr His Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Val Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu His Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Arg Leu Ala
Arg Gly Gly Pro Ser Ala His Ala Phe Glu 100
105 110Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
Ala Ser Thr Lys 115 120 125Gly Pro
Ser 130120111PRTArtificial SequenceSynthetically generated peptide
120Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45Ile Tyr Lys
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile
Ser Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly
Thr Gly Thr Lys Leu Thr Val Leu Ser 100 105
110121120PRTArtificial SequenceSynthetically generated
peptide 121Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Val Ile Ser Gly Asp Ser Val Ser Asn Asn 20
25 30Ser Ala Val Trp Asn Trp Ile Arg Gln Ser
Leu Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Ile Ile
Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val 85 90 95Tyr Tyr
Cys Ala Arg Arg Thr Gly Ala Gly Val Asp Tyr Trp Gly Gln 100
105 110Gly Thr Leu Val Thr Val Ser Ser
115 120122111PRTArtificial SequenceSynthetically
generated peptide 122Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr
Pro Gly Gln1 5 10 15Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20
25 30Tyr Val Tyr Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ala Ala
Ser Leu Ala Ile Ser Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95Arg
Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser 100
105 110123124PRTArtificial
SequenceSynthetically generated peptide 123Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Gly Val
Ser Gly Asn 20 25 30Asn Val
Ile Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Tyr Asp Leu Leu 50 55 60Pro
Ser Val Lys Ser Arg Ile Ala Ile Asn Pro Asp Thr Ser Lys Ser65
70 75 80Gln Phe Ser Leu Gln Leu
Ser Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Tyr Cys Ala Arg Thr Arg Ala Val Ala Gly Asn
Gln Tyr Phe Asp 100 105 110Leu
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115
120124111PRTArtificial SequenceSynthetically generated peptide 124Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Lys Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly Thr
Gly Thr Lys Leu Thr Val Leu Ser 100 105
110125129PRTArtificial SequenceSynthetically generated peptide
125Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Ser Leu
Ala Ala Ala Ala Gly Thr Val Asp Tyr Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser
Ala Ser Ala Pro 115 120
125Thr126111PRTArtificial SequenceSynthetically generated peptide 126Gln
Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys Ser
Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45Ile Tyr Lys Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser
Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly
Thr Gly Thr Lys Leu Thr Val Leu Ser 100 105
110127126PRTArtificial SequenceSynthetically generated
peptide 127Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20
25 30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser
Leu Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Leu Ser Val Lys Ser Arg Ile Asn Ile
Asn Ala Asp Thr Ser Lys Ser65 70 75
80Gln Phe Ser Leu Gln Leu Asp Ser Val Thr Pro Glu Asp Thr
Ala Val 85 90 95Tyr Phe
Cys Ala Lys Asp Arg Leu Leu Tyr Asn Tyr Gly Ser Asn Ala 100
105 110Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120
125128111PRTArtificial SequenceSynthetically generated peptide 128Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Lys Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly Thr
Gly Thr Lys Leu Thr Val Leu Ser 100 105
110129136PRTArtificial SequenceSynthetically generated peptide
129Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Asp
Thr Pro Arg Tyr Cys Ser Gly Gly Ser Cys 100
105 110Tyr Lys Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 115 120 125Ser Ala
Ser Thr Lys Ser Pro Ser 130 135130111PRTArtificial
SequenceSynthetically generated peptide 130Gln Pro Val Leu Thr Gln Ser
Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile
Gly Ser Asn 20 25 30Tyr Val
Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60Gly
Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly Leu Gln65
70 75 80Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 95Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr
Val Leu Ser 100 105
110131124PRTArtificial SequenceSynthetically generated peptide 131Gln Val
Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Ser Ser Gln1 5
10 15Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Lys 20 25
30Gly Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu
Glu 35 40 45Trp Leu Gly Arg Ala
Tyr Tyr Trp Ser Lys Trp Tyr Tyr Asp Tyr Ala 50 55
60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser
Lys Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Leu Thr Pro Glu Asp Thr Ala Val
85 90 95Phe Tyr Cys Arg Ala Thr Ser
Thr Tyr Tyr Leu Pro Gly Gly Leu Asp 100 105
110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120132111PRTArtificial SequenceSynthetically
generated peptide 132Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr
Pro Gly Gln1 5 10 15Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20
25 30Tyr Val Tyr Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ala Ala
Ser Leu Ala Ile Ser Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95Arg
Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser 100
105 110133121PRTArtificial
SequenceSynthetically generated peptide 133Gln Val Gln Leu Gln Gln Ser
Gly Ala Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Thr Ile Ser Gly Asp Ser Val
Ser Ala Asp 20 25 30Arg Val
Ala Trp Asn Trp Ile Arg Gln Ser Pro Leu Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Ile Phe Tyr Arg Ser Lys
Trp Met Val Asp Tyr Ala 50 55 60Val
Ser Val Lys Ser Arg Ile Ser Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu
Asn Ser Val Thr Pro Glu Asp Thr Ala Met 85
90 95Tyr Tyr Cys Ala Arg Ala Thr Thr Arg Gly Tyr Phe
Asp Leu Trp Gly 100 105 110Arg
Gly Thr Leu Val Thr Val Ser Ser 115
120134111PRTArtificial SequenceSynthetically generated peptide 134Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Lys Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly Thr
Gly Thr Lys Leu Thr Val Leu Ser 100 105
110135123PRTArtificial SequenceSynthetically generated peptide
135Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Val Val Ser Gly Asp Gly Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Met Lys Gly Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asp Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Arg
Ser Gly Arg Thr Gly Gly Tyr Phe Asp Leu 100
105 110Trp Gly Arg Gly Thr Leu Val Ala Val Ser Ser
115 120136111PRTArtificial SequenceSynthetically
generated peptide 136Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr
Pro Gly Gln1 5 10 15Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20
25 30Tyr Val Tyr Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ala Ala
Ser Leu Ala Ile Ser Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95Arg
Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser 100
105 110137120PRTArtificial
SequenceSynthetically generated peptide 137Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Pro Gly His Ser Val
Gly Ser Ser 20 25 30Asn Ala
Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Ile Phe Tyr Gly Ser Lys
Trp Tyr Asn Asp Tyr Ala 50 55 60Val
Ser Leu Lys Ser Arg Leu Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu
Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Tyr Cys Ala Arg Arg Thr Gly Thr Gly Ile Asp
Tyr Trp Gly Gln 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115
120138111PRTArtificial SequenceSynthetically generated peptide 138Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Lys Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly Thr
Gly Thr Lys Leu Thr Val Leu Ser 100 105
110139118PRTArtificial SequenceSynthetically generated peptide
139Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly His Ser Val Gly Ser Ser 20 25
30Asn Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Ile Phe Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Thr Arg Ile Ser Ile Asn Pro Asp
Thr Ala Lys Asn65 70 75
80Gln Phe Ser Leu His Leu Asn Ser Val Thr Ala Glu Asp Thr Gly Val
85 90 95Tyr Tyr Cys Arg Gln Gln
Lys Arg Leu Asp Ser Trp Gly Gln Gly Thr 100
105 110Leu Val Thr Val Ser Ser
115140111PRTArtificial SequenceSynthetically generated peptide 140Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Lys Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly Thr
Gly Thr Lys Leu Thr Val Leu Ser 100 105
110141132PRTArtificial SequenceSynthetically generated peptide
141Pro Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Pro Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Asp Asp Thr Ala Ile
85 90 95Tyr Tyr Cys Ala Arg Ala
Ser Asp Tyr Gly Asp Tyr Phe Tyr Tyr Phe 100
105 110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Ser Ala 115 120 125Ser Ala
Pro Thr 130142111PRTArtificial SequenceSynthetically generated peptide
142Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45Ile Tyr Lys
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile
Ser Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly
Thr Gly Thr Lys Leu Thr Val Leu Ser 100 105
110143125PRTArtificial SequenceSynthetically generated
peptide 143Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20
25 30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser
Pro Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Ile Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Ala Ile
Lys Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val 85 90 95Tyr Tyr
Cys Arg Ala Gly Arg Ser Phe Asp Leu Trp Gly Arg Gly Thr 100
105 110Leu Val Thr Val Ser Ser Gly Ser Ala
Ser Ala Pro Thr 115 120
125144111PRTArtificial SequenceSynthetically generated peptide 144Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Lys Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly Thr
Gly Thr Lys Leu Thr Val Leu Ser 100 105
110145128PRTArtificial SequenceSynthetically generated peptide
145Gln Val Gln Leu Gln Gln Ser Ser Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Val Ser Gly Asp Ser Val Ser Gly Asn 20 25
30Ser Gly Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Tyr Thr Tyr Lys Trp Tyr Ile Asp Tyr 50
55 60Ala Val Ser Val Lys Ser Arg Ile Thr Val Asn Pro
Asp Thr Ser Arg65 70 75
80Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala
85 90 95Val Tyr Tyr Cys Ala Arg
Val Asp Tyr Thr Gly Ser Pro Val Trp Gly 100
105 110Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ala
Ser Ala Pro Thr 115 120
125146111PRTArtificial SequenceSynthetically generated peptide 146Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Lys Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly Thr
Gly Thr Lys Leu Thr Val Leu Ser 100 105
110147132PRTArtificial SequenceSynthetically generated peptide
147Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Arg Gly Arg Ile Thr Ile Asn Ala Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr
Gly Tyr Ser Ser Ser Trp Val Val Asn Phe 100
105 110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Ser Ala 115 120 125Ser Ala
Pro Thr 130148111PRTArtificial SequenceSynthetically generated peptide
148Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45Ile Tyr Lys
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile
Ser Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Arg Gly Tyr Val Phe Gly
Thr Gly Thr Lys Leu Thr Val Leu Ser 100 105
110149265PRTArtificial SequenceSynthetically generated
peptide 149Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Ile 20
25 30Thr Ala Ala Trp Asn Trp Leu Arg Gln Ser
Pro Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr His Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Val
Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu His Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val 85 90 95Tyr Tyr
Cys Arg Leu Ala Arg Gly Gly Pro Ser Ala His Ala Phe Glu 100
105 110Ile Trp Gly Gln Gly Thr Met Val Thr
Val Ser Ser Ala Ser Thr Lys 115 120
125Gly Pro Ser Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140Gly Ser Gly Gly Gly Gly Ser
Gln Pro Val Leu Thr Gln Ser Pro Ser145 150
155 160Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser
Cys Ser Gly Ser 165 170
175 Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro
180 185 190Gly Thr Ala Pro Lys Leu
Leu Ile Tyr Lys Asn Asn Gln Arg Pro Ser 195 200
205Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ala
Ala Ser 210 215 220Leu Ala Ile Ser Gly
Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys225 230
235 240Ala Ala Trp Asp Asp Ser Leu Arg Gly Tyr
Val Phe Gly Thr Gly Thr 245 250
255Lys Leu Thr Val Leu Ser Gly Ile Leu 260
265150254PRTArtificial SequenceSynthetically generated peptide 150Gln
Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Val
Ile Ser Gly Asp Ser Val Ser Asn Asn 20 25
30Ser Ala Val Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Lys Ser Arg Ile Ile Ile Asn Pro Asp Thr
Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Arg
Thr Gly Ala Gly Val Asp Tyr Trp Gly Gln 100
105 110Gly Thr Leu Val Thr Val Ser Ser Gly Ile Leu Gly
Ser Gly Gly Gly 115 120 125Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val Leu 130
135 140Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly
Gln Arg Val Thr Ile145 150 155
160Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp
165 170 175Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys Asn 180
185 190Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Lys Ser 195 200 205Gly
Thr Ala Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu 210
215 220Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Ser Leu Arg Gly Tyr Val225 230 235
240Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser Gly Ile Leu
245 250151258PRTArtificial SequenceSynthetically
generated peptide 151Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Gly Val Ser Gly Asn 20
25 30Asn Val Ile Trp Asn Trp Ile Arg
Gln Ser Pro Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Leu Leu
50 55 60Pro Ser Val Lys Ser Arg Ile Ala
Ile Asn Pro Asp Thr Ser Lys Ser65 70 75
80Gln Phe Ser Leu Gln Leu Ser Ser Val Thr Pro Glu Asp
Thr Ala Val 85 90 95Tyr
Tyr Cys Ala Arg Thr Arg Ala Val Ala Gly Asn Gln Tyr Phe Asp
100 105 110Leu Trp Gly Arg Gly Thr Leu
Val Thr Val Ser Ser Gly Ile Leu Gly 115 120
125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 130 135 140Gln Pro Val Leu Thr Gln
Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln145 150
155 160Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser
Asn Ile Gly Ser Asn 165 170
175Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
180 185 190Ile Tyr Lys Asn Asn Gln
Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 195 200
205Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln 210 215 220Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu225 230
235 240Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys
Leu Thr Val Leu Ser Gly 245 250
255Ile Leu152263PRTArtificial SequenceSynthetically generated
peptide 152Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr 20
25 30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser
Leu Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Ile
Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val 85 90 95Tyr Tyr
Cys Ala Ser Leu Ala Ala Ala Ala Gly Thr Val Asp Tyr Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Ser Ala Ser Ala Pro 115 120
125Thr Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140Gly Gly Gly Gly Ser Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser145 150
155 160Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser
Gly Ser Ser Ser 165 170
175Asn Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr
180 185 190Ala Pro Lys Leu Leu Ile
Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val 195 200
205Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ala Ala Ser
Leu Ala 210 215 220Ile Ser Gly Leu Gln
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala225 230
235 240Trp Asp Asp Ser Leu Arg Gly Tyr Val Phe
Gly Thr Gly Thr Lys Leu 245 250
255Thr Val Leu Ser Gly Ile Leu 260153260PRTArtificial
SequenceSynthetically generated peptide 153Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val
Ser Ser Asn 20 25 30Ser Ala
Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Asn Asp Tyr Ala 50 55 60Leu
Ser Val Lys Ser Arg Ile Asn Ile Asn Ala Asp Thr Ser Lys Ser65
70 75 80Gln Phe Ser Leu Gln Leu
Asp Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Phe Cys Ala Lys Asp Arg Leu Leu Tyr Asn Tyr
Gly Ser Asn Ala 100 105 110Met
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ile 115
120 125Leu Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly 130 135
140Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro145
150 155 160Gly Gln Arg Val
Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly 165
170 175Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys 180 185
190Leu Leu Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
195 200 205Phe Ser Gly Ser Lys Ser Gly
Thr Ala Ala Ser Leu Ala Ile Ser Gly 210 215
220Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp225 230 235 240Ser Leu
Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu
245 250 255Ser Gly Ile Leu
260154270PRTArtificial SequenceSynthetically generated peptide 154Gln Val
Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5
10 15Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg Gly Leu
Glu 35 40 45Trp Leu Gly Arg Thr
Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser
Lys Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Asp Thr
Pro Arg Tyr Cys Ser Gly Gly Ser Cys 100 105
110Tyr Lys Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser 115 120 125Ser Ala Ser Thr
Lys Ser Pro Ser Gly Ile Leu Gly Ser Gly Gly Gly 130
135 140Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gln Pro Val Leu145 150 155
160Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile
165 170 175Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp 180
185 190Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
Ile Tyr Lys Asn 195 200 205Asn Gln
Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser 210
215 220Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly Leu
Gln Ser Glu Asp Glu225 230 235
240Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Arg Gly Tyr Val
245 250 255Phe Gly Thr Gly
Thr Lys Leu Thr Val Leu Ser Gly Ile Leu 260
265 270155258PRTArtificial SequenceSynthetically
generated peptide 155Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys
Ser Ser Gln1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Lys 20
25 30Gly Ala Ala Trp Asn Trp Ile Arg
Gln Ser Pro Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Ala Tyr Tyr Trp Ser Lys Trp Tyr Tyr Asp Tyr Ala
50 55 60Val Ser Val Lys Ser Arg Ile Thr
Ile Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Leu Thr Pro Glu Asp
Thr Ala Val 85 90 95Phe
Tyr Cys Arg Ala Thr Ser Thr Tyr Tyr Leu Pro Gly Gly Leu Asp
100 105 110Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser Gly Ile Leu Gly 115 120
125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 130 135 140Gln Pro Val Leu Thr Gln
Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln145 150
155 160Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser
Asn Ile Gly Ser Asn 165 170
175Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
180 185 190Ile Tyr Lys Asn Asn Gln
Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 195 200
205Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly
Leu Gln 210 215 220Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu225 230
235 240Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys
Leu Thr Val Leu Ser Gly 245 250
255Ile Leu156255PRTArtificial SequenceSynthetically generated
peptide 156Gln Val Gln Leu Gln Gln Ser Gly Ala Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Ile Ser Gly Asp Ser Val Ser Ala Asp 20
25 30Arg Val Ala Trp Asn Trp Ile Arg Gln Ser
Pro Leu Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Ile Phe Tyr Arg Ser Lys Trp Met Val Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Ser Ile
Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Met 85 90 95Tyr Tyr
Cys Ala Arg Ala Thr Thr Arg Gly Tyr Phe Asp Leu Trp Gly 100
105 110Arg Gly Thr Leu Val Thr Val Ser Ser
Gly Ile Leu Gly Ser Gly Gly 115 120
125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val
130 135 140Leu Thr Gln Ser Pro Ser Ala
Ser Gly Thr Pro Gly Gln Arg Val Thr145 150
155 160Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser
Asn Tyr Val Tyr 165 170
175Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys
180 185 190Asn Asn Gln Arg Pro Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Lys 195 200
205Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser
Glu Asp 210 215 220Glu Ala Asp Tyr Tyr
Cys Ala Ala Trp Asp Asp Ser Leu Arg Gly Tyr225 230
235 240Val Phe Gly Thr Gly Thr Lys Leu Thr Val
Leu Ser Gly Ile Leu 245 250
255157257PRTArtificial SequenceSynthetically generated peptide 157Gln
Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Val
Val Ser Gly Asp Gly Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Met Lys Gly Arg Ile Thr Ile Asn Pro Asp Thr
Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asp Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95 Tyr Tyr Cys Ala Arg Arg
Ser Gly Arg Thr Gly Gly Tyr Phe Asp Leu 100
105 110Trp Gly Arg Gly Thr Leu Val Ala Val Ser Ser Gly
Ile Leu Gly Ser 115 120 125Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130
135 140Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly
Thr Pro Gly Gln Arg145 150 155
160Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr
165 170 175Val Tyr Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 180
185 190Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser Gly 195 200 205Ser
Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser 210
215 220Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala
Trp Asp Asp Ser Leu Arg225 230 235
240Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser Gly
Ile 245 250
255Leu158254PRTArtificial SequenceSynthetically generated peptide 158Gln
Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala
Ile Pro Gly His Ser Val Gly Ser Ser 20 25
30Asn Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg
Ile Phe Tyr Gly Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Leu Lys Ser Arg Leu Thr Ile Asn Pro Asp Thr
Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Arg
Thr Gly Thr Gly Ile Asp Tyr Trp Gly Gln 100
105 110Gly Thr Leu Val Thr Val Ser Ser Gly Ile Leu Gly
Ser Gly Gly Gly 115 120 125Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val Leu 130
135 140Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly
Gln Arg Val Thr Ile145 150 155
160Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr Trp
165 170 175Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys Asn 180
185 190Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Lys Ser 195 200 205Gly
Thr Ala Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu 210
215 220Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Ser Leu Arg Gly Tyr Val225 230 235
240Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser Gly Ile Leu
245 250159252PRTArtificial SequenceSynthetically
generated peptide 159Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Ile Ser Gly His Ser Val Gly Ser Ser 20
25 30Asn Ala Ala Trp Asn Trp Ile Arg
Gln Ser Pro Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Ile Phe Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60Val Ser Val Lys Thr Arg Ile Ser
Ile Asn Pro Asp Thr Ala Lys Asn65 70 75
80Gln Phe Ser Leu His Leu Asn Ser Val Thr Ala Glu Asp
Thr Gly Val 85 90 95Tyr
Tyr Cys Arg Gln Gln Lys Arg Leu Asp Ser Trp Gly Gln Gly Thr
100 105 110Leu Val Thr Val Ser Ser Gly
Ile Leu Gly Ser Gly Gly Gly Gly Ser 115 120
125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val Leu Thr
Gln 130 135 140Ser Pro Ser Ala Ser Gly
Thr Pro Gly Gln Arg Val Thr Ile Ser Cys145 150
155 160Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr
Val Tyr Trp Tyr Gln 165 170
175Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys Asn Asn Gln
180 185 190Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr 195 200
205Ala Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu
Ala Asp 210 215 220Tyr Tyr Cys Ala Ala
Trp Asp Asp Ser Leu Arg Gly Tyr Val Phe Gly225 230
235 240Thr Gly Thr Lys Leu Thr Val Leu Ser Gly
Ile Leu 245 250160266PRTArtificial
SequenceSynthetically generated peptide 160Pro Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Pro Cys Ala Ile Ser Gly Asp Ser Val
Ser Ser Asn 20 25 30Ser Ala
Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Asn Asp Tyr Ala 50 55 60Val
Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu
Asn Ser Val Thr Pro Asp Asp Thr Ala Ile 85
90 95Tyr Tyr Cys Ala Arg Ala Ser Asp Tyr Gly Asp Tyr
Phe Tyr Tyr Phe 100 105 110Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ala 115
120 125Ser Ala Pro Thr Gly Ile Leu Gly Ser
Gly Gly Gly Gly Ser Gly Gly 130 135
140Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Pro145
150 155 160Ser Ala Ser Gly
Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly 165
170 175Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val
Tyr Trp Tyr Gln Gln Leu 180 185
190Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys Asn Asn Gln Arg Pro
195 200 205Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Lys Ser Gly Thr Ala Ala 210 215
220Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr
Tyr225 230 235 240Cys Ala
Ala Trp Asp Asp Ser Leu Arg Gly Tyr Val Phe Gly Thr Gly
245 250 255Thr Lys Leu Thr Val Leu Ser
Gly Ile Leu 260 265161259PRTArtificial
SequenceSynthetically generated peptide 161Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val
Ser Ser Asn 20 25 30Ser Ala
Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Ile Tyr Tyr Arg Ser Lys
Trp Tyr Tyr Asp Tyr Ala 50 55 60Val
Ser Val Lys Ser Arg Ile Ala Ile Lys Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu
Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Tyr Cys Arg Ala Gly Arg Ser Phe Asp Leu Trp
Gly Arg Gly Thr 100 105 110Leu
Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr Gly Ile Leu 115
120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 130 135
140Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly145
150 155 160Gln Arg Val Thr
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser 165
170 175Asn Tyr Val Tyr Trp Tyr Gln Gln Leu Pro
Gly Thr Ala Pro Lys Leu 180 185
190Leu Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe
195 200 205Ser Gly Ser Lys Ser Gly Thr
Ala Ala Ser Leu Ala Ile Ser Gly Leu 210 215
220Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Ser225 230 235 240Leu Arg
Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser
245 250 255Gly Ile Leu162262PRTArtificial
SequenceSynthetically generated peptide 162Gln Val Gln Leu Gln Gln Ser
Ser Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Asp Ser Val
Ser Gly Asn 20 25 30Ser Gly
Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Tyr Thr Tyr
Lys Trp Tyr Ile Asp Tyr 50 55 60Ala
Val Ser Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Arg65
70 75 80Asn Gln Phe Ser Leu Gln
Leu Asn Ser Val Thr Pro Glu Asp Thr Ala 85
90 95Val Tyr Tyr Cys Ala Arg Val Asp Tyr Thr Gly Ser
Pro Val Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr 115
120 125Gly Ile Leu Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly 130 135
140Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly145
150 155 160Thr Pro Gly Gln
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn 165
170 175Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln
Gln Leu Pro Gly Thr Ala 180 185
190Pro Lys Leu Leu Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro
195 200 205Asp Arg Phe Ser Gly Ser Lys
Ser Gly Thr Ala Ala Ser Leu Ala Ile 210 215
220Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala
Trp225 230 235 240Asp Asp
Ser Leu Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr
245 250 255Val Leu Ser Gly Ile Leu
260163266PRTArtificial SequenceSynthetically generated peptide 163Gln
Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala
Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Leu Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Arg Gly Arg Ile Thr Ile Asn Ala Asp Thr
Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr
Gly Tyr Ser Ser Ser Trp Val Val Asn Phe 100
105 110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Ser Ala 115 120 125Ser Ala
Pro Thr Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly 130
135 140Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val
Leu Thr Gln Ser Pro145 150 155
160Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly
165 170 175Ser Ser Ser Asn
Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu 180
185 190Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys
Asn Asn Gln Arg Pro 195 200 205Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ala Ala 210
215 220Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu
Asp Glu Ala Asp Tyr Tyr225 230 235
240Cys Ala Ala Trp Asp Asp Ser Leu Arg Gly Tyr Val Phe Gly Thr
Gly 245 250 255Thr Lys Leu
Thr Val Leu Ser Gly Ile Leu 260
2651647PRTArtificial SequenceSynthetically generated peptide 164Ser Ile
Thr Ala Ala Trp Asn1 516518PRTArtificial
SequenceSynthetically generated peptide 165Arg Thr Tyr His Arg Ser Lys
Trp Tyr Tyr Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser16616PRTArtificial SequenceSynthetically
generated peptide 166Gly Leu Ala Ala Arg Gly Gly Gly Pro Ser Ala His Ala
Phe Glu Ile1 5 10
151678PRTArtificial SequenceSynthetically generated peptide 167Ser Ser
Ile Thr Ala Ala Trp Asn1 516819PRTArtificial
SequenceSynthetically generated peptide 168Arg Thr Tyr His Arg Ser Lys
Trp Tyr Tyr Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser Arg16916PRTArtificial SequenceSynthetically
generated peptide 169Gly Leu Ala Ala Arg Gly Gly Gly Pro Ser Ala His Ala
Phe Glu Ile1 5 10
151707PRTArtificial SequenceSynthetically generated peptide 170Asn Asn
Ser Ala Val Trp Asn1 51718PRTArtificial
SequenceSynthetically generated peptide 171Ser Asn Asn Ser Ala Val Trp
Asn1 51728PRTArtificial SequenceSynthetically generated
peptide 172Arg Thr Gly Ala Gly Val Asp Tyr1
51738PRTArtificial SequenceSynthetically generated peptide 173Ser Gly Asn
Asn Val Ile Trp Asn1 517419PRTArtificial
SequenceSynthetically generated peptide 174Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Tyr Asp Leu Leu Pro Ser Val1 5 10
15Lys Ser Arg17512PRTArtificial SequenceSynthetically
generated peptide 175Thr Arg Ala Val Ala Gly Asn Gln Tyr Phe Asp Leu1
5 101767PRTArtificial SequenceSynthetically
generated peptide 176Gly Asn Asn Val Ile Trp Asn1
517718PRTArtificial SequenceSynthetically generated peptide 177Arg Thr
Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Leu Leu Pro Ser Val1 5
10 15Lys Ser17812PRTArtificial
SequenceSynthetically generated peptide 178Thr Arg Ala Val Ala Gly Asn
Gln Tyr Phe Asp Leu1 5 101798PRTArtificial
SequenceSynthetically generated peptide 179Ser Ser Tyr Ser Ala Ala Trp
Asn1 518019PRTArtificial SequenceSynthetically generated
peptide 180Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser
Val1 5 10 15Lys Ser
Arg18110PRTArtificial SequenceSynthetically generated peptide 181Leu Ala
Ala Ala Ala Gly Thr Val Asp Tyr1 5
101827PRTArtificial SequenceSynthetically generated peptide 182Ser Tyr
Ser Ala Ala Trp Asn1 51838PRTArtificial
SequenceSynthetically generated peptide 183Ser Ser Asn Ser Ala Ala Trp
Asn1 518410PRTArtificial SequenceSynthetically generated
peptide 184Leu Ala Ala Ala Ala Gly Thr Val Asp Tyr1 5
1018519PRTArtificial SequenceSynthetically generated peptide
185Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Leu Ser Val1
5 10 15Lys Ser
Arg18613PRTArtificial SequenceSynthetically generated peptide 186Asp Arg
Leu Leu Tyr Asn Tyr Gly Ser Asn Ala Met Asp1 5
101878PRTArtificial SequenceSynthetically generated peptide 187Ser
Ser Asn Ser Ala Ala Trp Asn1 518819PRTArtificial
SequenceSynthetically generated peptide 188Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser Arg18918PRTArtificial SequenceSynthetically
generated peptide 189Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
Leu Ser Val1 5 10 15Lys
Ser19014PRTArtificial SequenceSynthetically generated peptide 190Asp Arg
Leu Leu Tyr Asn Tyr Gly Ser Asn Ala Met Asp Val1 5
1019116PRTArtificial SequenceSynthetically generated peptide
191Asp Thr Pro Arg Tyr Cys Ser Gly Gly Ser Cys Tyr Lys Tyr Phe Asp1
5 10 151928PRTArtificial
SequenceSynthetically generated peptide 192Ser Ser Lys Gly Ala Ala Trp
Asn1 519319PRTArtificial SequenceSynthetically generated
peptide 193Arg Ala Tyr Tyr Trp Ser Lys Trp Tyr Tyr Asp Tyr Ala Val Ser
Val1 5 10 15Lys Ser
Arg19414PRTArtificial SequenceSynthetically generated peptide 194Gly Ala
Thr Ser Thr Tyr Tyr Leu Pro Gly Gly Leu Asp Val1 5
101958PRTArtificial SequenceSynthetically generated peptide
195Ser Ala Asp Arg Val Ala Trp Asn1 519617PRTArtificial
SequenceSynthetically generated peptide 196Asp Thr Pro Arg Tyr Cys Ser
Gly Gly Ser Cys Tyr Lys Tyr Phe Asp1 5 10
15Tyr19719PRTArtificial SequenceSynthetically generated
peptide 197Arg Ile Phe Tyr Arg Ser Lys Trp Met Val Asp Tyr Ala Val Ser
Val1 5 10 15Lys Ser
Arg1989PRTArtificial SequenceSynthetically generated peptide 198Ala Thr
Thr Arg Gly Tyr Phe Asp Leu1 519919PRTArtificial
SequenceSynthetically generated peptide 199Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Asn Asp Tyr Ala Val Ser Met1 5 10
15Lys Gly Arg2007PRTArtificial SequenceSynthetically
generated peptide 200Ser Lys Gly Ala Ala Trp Asn1
520118PRTArtificial SequenceSynthetically generated peptide 201Arg Ala
Tyr Tyr Trp Ser Lys Trp Tyr Tyr Asp Tyr Ala Val Ser Val1 5
10 15Lys Ser20214PRTArtificial
SequenceSynthetically generated peptide 202Gly Ala Thr Ser Thr Tyr Tyr
Leu Pro Gly Gly Leu Asp Val1 5
1020311PRTArtificial SequenceSynthetically generated peptide 203Arg Ser
Gly Arg Thr Gly Gly Tyr Phe Asp Leu1 5
102048PRTArtificial SequenceSynthetically generated peptide 204Gly Ser
Ser Asn Ala Ala Trp Asn1 520519PRTArtificial
SequenceSynthetically generated peptide 205Arg Ile Phe Tyr Gly Ser Lys
Trp Tyr Asn Asp Tyr Ala Val Ser Leu1 5 10
15Lys Ser Arg2067PRTArtificial SequenceSynthetically
generated peptide 206Ala Asp Arg Val Ala Trp Asn1
520718PRTArtificial SequenceSynthetically generated peptide 207Arg Ile
Phe Tyr Arg Ser Lys Trp Met Val Asp Tyr Ala Val Ser Val1 5
10 15Lys Ser2089PRTArtificial
SequenceSynthetically generated peptide 208Ala Thr Thr Arg Gly Tyr Phe
Asp Leu1 52098PRTArtificial SequenceSynthetically generated
peptide 209Arg Thr Gly Thr Gly Ile Asp Tyr1
521019PRTArtificial SequenceSynthetically generated peptide 210Arg Ile
Phe Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5
10 15Lys Thr Arg2118PRTArtificial
SequenceSynthetically generated peptide 211Gly Gln Gln Lys Arg Leu Asp
Ser1 521213PRTArtificial SequenceSynthetically generated
peptide 212Thr Gly Tyr Ser Ser Ser Trp Val Val Asn Phe Asp Tyr1
5 1021318PRTArtificial SequenceSynthetically
generated peptide 213Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
Val Ser Met1 5 10 15Lys
Gly21411PRTArtificial SequenceSynthetically generated peptide 214Arg Ser
Gly Arg Thr Gly Gly Tyr Phe Asp Leu1 5
1021513PRTArtificial SequenceSynthetically generated peptide 215Ala Ser
Asp Tyr Gly Asp Tyr Phe Tyr Tyr Phe Asp Tyr1 5
1021619PRTArtificial SequenceSynthetically generated peptide 216Arg
Ile Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala Val Ser Val1
5 10 15Lys Ser Arg2178PRTArtificial
SequenceSynthetically generated peptide 217Gly Ala Gly Arg Ser Phe Asp
Leu1 52187PRTArtificial SequenceSynthetically generated
peptide 218Ser Ser Asn Ala Ala Trp Asn1 521918PRTArtificial
SequenceSynthetically generated peptide 219Arg Ile Phe Tyr Gly Ser Lys
Trp Tyr Asn Asp Tyr Ala Val Ser Leu1 5 10
15Lys Ser2208PRTArtificial SequenceSynthetically
generated peptide 220Arg Thr Gly Thr Gly Ile Asp Tyr1
52218PRTArtificial SequenceSynthetically generated peptide 221Ser Gly Asn
Ser Gly Val Trp Asn1 522220PRTArtificial
SequenceSynthetically generated peptide 222Arg Thr Tyr Tyr Tyr Thr Tyr
Lys Trp Tyr Ile Asp Tyr Ala Val Ser1 5 10
15Val Lys Ser Arg 202238PRTArtificial
SequenceSynthetically generated peptide 223Val Asp Tyr Thr Gly Ser Pro
Val1 52247PRTArtificial SequenceSynthetically generated
peptide 224Ser Ser Asn Ala Ala Trp Asn1 522518PRTArtificial
SequenceSynthetically generated peptide 225Arg Ile Phe Tyr Arg Ser Lys
Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5 10
15Lys Thr2268PRTArtificial SequenceSynthetically
generated peptide 226Gly Gln Gln Lys Arg Leu Asp Ser1
522719PRTArtificial SequenceSynthetically generated peptide 227Arg Thr
Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5
10 15Arg Gly Arg22813PRTArtificial
SequenceSynthetically generated peptide 228Thr Gly Tyr Ser Ser Ser Trp
Val Val Asn Phe Asp Tyr1 5
1022910PRTArtificial SequenceSynthetically generated peptide 229Ser Gln
Xaa Val Ser Xaa Xaa Xaa Leu Ala1 5
102307PRTArtificial SequenceSynthetically generated peptide 230Asp Val
Ala Asn Xaa Ala Ala1 52318PRTArtificial
SequenceSynthetically generated peptide 231Gln Gln Arg Ser Gln Trp Pro
Gln1 523213PRTArtificial SequenceSynthetically generated
peptide 232Ala Ser Asp Tyr Gly Asp Tyr Phe Tyr Tyr Phe Asp Tyr1
5 102334PRTArtificial SequenceSynthetically
generated peptide 233Xaa Ala Met Xaa12347PRTArtificial
SequenceSynthetically generated peptide 234Tyr Ala Xaa Ser Val Lys Xaa1
523513PRTArtificial SequenceSynthetically generated peptide
235Ser Gly Ser Ser Ser Asn Ile Xaa Ser Asn Xaa Val Xaa1 5
102367PRTArtificial SequenceSynthetically generated
peptide 236Xaa Xaa Xaa Ala Ala Trp Xaa1 523718PRTArtificial
SequenceSynthetically generated peptide 237Arg Ile Tyr Tyr Arg Ser Lys
Trp Tyr Tyr Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser2388PRTArtificial SequenceSynthetically
generated peptide 238Gly Ala Gly Arg Ser Phe Asp Leu1
523918PRTArtificial SequenceSynthetically generated peptide 239Arg Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Ala Xaa Xaa Xaa1 5
10 15Lys Ser2407PRTArtificial
SequenceSynthetically generated peptide 240Asp Val Xaa Xaa Arg Pro Ser1
524111PRTArtificial SequenceSynthetically generated peptide
241Leu Xaa Xaa Xaa Xaa Xaa Asn Gln Arg Pro Ser1 5
102428PRTArtificial SequenceSynthetically generated peptide
242Gly Asn Ser Gly Val Trp Asn Trp1 524318PRTArtificial
SequenceSynthetically generated peptide 243Thr Tyr Tyr Tyr Thr Tyr Lys
Trp Tyr Ile Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser2448PRTArtificial SequenceSynthetically
generated peptide 244Val Asp Tyr Thr Gly Ser Pro Val1
52455PRTArtificial SequenceSynthetically generated peptide 245Ala Trp Asp
Asp Ser1 52469PRTArtificial SequenceSynthetically generated
peptide 246Ser Ser Ser Trp Val Val Xaa Phe Xaa1
524729PRTArtificial SequenceSynthetically generated peptide 247Cys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa1 5
10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Cys Xaa Cys 20 2524819PRTArtificial
SequenceSynthetically generated peptide 248Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser Arg2498PRTArtificial SequenceSynthetically
generated peptide 249Ser Ser Asn Ser Ala Ala Trp Asn1
52508PRTArtificial SequenceSynthetically generated peptide 250Gly Ser Ser
Asn Ala Ala Trp Asn1 525119PRTArtificial
SequenceSynthetically generated peptide 251Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser Arg2528PRTArtificial SequenceSynthetically
generated peptide 252Ser Ser Asn Ser Ala Ala Trp Asn1
52538PRTArtificial SequenceSynthetically generated peptide 253Ser Ser Asn
Ser Ala Ala Trp Asn1 5254121PRTArtificial
SequenceSynthetically generated peptide 254Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val
Ser Ser Asn 20 25 30Ser Ala
Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Thr Lys
Trp Tyr Asn Glu Tyr Ala 50 55 60Ala
Ser Val Lys Gly Arg Ala Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu
Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Tyr Cys Ala Thr Asp Pro Lys Gly Val Thr Thr
Gln Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser 115
120255110PRTArtificial SequenceSynthetically generated peptide 255Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly
Ser Thr Ser Asn Ile Gly Arg Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Arg Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Arg Asn Asn
Gln Arg Pro Ser Gly Ala Pro Ala Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ser Thr Ser Leu Ala Ile Ser Gly
Leu Arg65 70 75 80Ser
Glu Asp Glu Ala Glu Tyr Phe Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Ser Gly Trp Val Phe Gly Gly
Gly Thr Gln Leu Thr Val Leu 100 105
110256124PRTArtificial SequenceSynthetically generated peptide
256Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Gly Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Arg Trp Tyr Asn Asp Tyr Ala 50
55 60Ala Ser Val Lys Ser Arg Ile Thr Val Asn Ala Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Ser
Val Arg Tyr Ser Ser Gly Trp Gly Phe Asp 100
105 110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120257111PRTArtificial SequenceSynthetically
generated peptide 257Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr
Pro Gly Gln1 5 10 15Arg
Ile Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20
25 30Tyr Val Tyr Trp Tyr Gln Gln Phe
Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Val Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95Ser
Gly Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110258124PRTArtificial
SequenceSynthetically generated peptide 258Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val
Ser Arg Asn 20 25 30Ser Ala
Ala Trp Asn Leu Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Thr Asp Tyr Ala 50 55 60Val
Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Arg Leu
Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Tyr Cys Ala Arg Ser Gly Gly Gly His Ala Ala
Gly Lys Phe Asp 100 105 110Ser
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115
120259110PRTArtificial SequenceSynthetically generated peptide 259Gln Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Pro Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Lys 20 25
30Tyr Val Tyr Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45Ile Tyr Arg Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu Arg65 70 75 80Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu
85 90 95Ser Ala Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105
110260120PRTArtificial SequenceSynthetically generated peptide
260Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Asp Asp Arg Ala Val
85 90 95Tyr Tyr Cys Arg Gly Arg
Leu Gly Gly Gly Met Asp Val Trp Gly Gln 100
105 110Gly Thr Thr Val Thr Val Ser Ser 115
120261108PRTArtificial SequenceSynthetically generated peptide
261Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile Asn Tyr Val 20 25
30Tyr Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu
Leu Ile Tyr 35 40 45Thr Asn Asn
Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50
55 60Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu Arg Ser Glu65 70 75
80Asp Glu Ala Asp Tyr Phe Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly
85 90 95Trp Val Phe Gly Gly Gly
Thr Lys Val Thr Val Leu 100
105262121PRTArtificial SequenceSynthetically generated peptide 262Gln Val
Gln Leu Val Gln Ser Glu Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Gly Val
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Ser Tyr
Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Val Thr Thr Gly
Ile Thr Arg Tyr Phe Asp Leu Trp Gly 100 105
110Arg Gly Thr Leu Val Thr Val Ser Ser 115
120263111PRTArtificial SequenceSynthetically generated peptide
263Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1
5 10 15Ser Ile Thr Ile Ser Cys
Thr Gly Thr Asn Ser Asp Ile Gly Gly Tyr 20 25
30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45Met Ile Phe
Glu Val Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50
55 60Ser Ala Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Ile Ser Gly Leu65 70 75
80Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser
85 90 95Asn Thr Pro Ser Val Phe
Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105
110264120PRTArtificial SequenceSynthetically generated
peptide 264Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20
25 30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser
Pro Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Gly Arg Ile Thr Ile
Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Met 85 90 95Tyr Tyr
Cys Val Arg Ser Gly Gly Gly Arg Val Asp Pro Trp Gly Gln 100
105 110Gly Thr Leu Val Thr Val Ser Ser
115 120265110PRTArtificial SequenceSynthetically
generated peptide 265Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr
Pro Gly Gln1 5 10 15Arg
Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20
25 30Tyr Val Tyr Trp Tyr Gln Gln Leu
Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Arg Asn His Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60Ala Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95Ser
Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100
105 110266119PRTArtificial SequenceSynthetically
generated peptide 266Gln Val Gln Leu Val Gln Ser Glu Gly Gly Val Val Gln
Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Thr Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Leu
Tyr Tyr Cys 85 90 95Ala
Arg Glu Ala Ser Ser Gly Trp Tyr Ile Asp Ser Trp Gly Gln Gly
100 105 110Thr Leu Val Thr Val Ser Ser
115267109PRTArtificial SequenceSynthetically generated peptide 267Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln
85 90 95Val Thr Phe Gly Gln Gly
Thr Arg Leu Glu Val Lys Ser 100
105268125PRTArtificial SequenceSynthetically generated peptide 268Arg Val
Gln Leu Gln Gln Leu Gly Pro Gly Leu Val Lys Pro Ser Gln1 5
10 15Thr Leu Ser Leu Thr Cys Ala Ile
Phe Gly Asp Ser Val Ser Ser Asn 20 25
30Gly Ala Ala Trp Ser Trp Ile Arg Gln Ser Leu Ser Arg Gly Leu
Glu 35 40 45Trp Leu Gly Arg Ala
Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Arg Gly Arg Ile Thr Ile Asn Ala Asp Thr Ser
Lys Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr Gly
Tyr Ser Ser Ser Arg Val Val Ser Ser 100 105
110Gly Tyr Trp Gly Gln Gly Thr Leu Val Ala Val Ser Ser
115 120 125269110PRTArtificial
SequenceSynthetically generated peptide 269Gln Pro Val Leu Thr Gln Ser
Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile
Gly Ser Asn 20 25 30Tyr Val
Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45Ile Tyr Lys Ser Asn Arg Arg Pro Ser Gly
Val Pro Gly Arg Phe Ser 50 55 60Gly
Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly Leu Gln65
70 75 80Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 95Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr
Val Leu 100 105
110270125PRTArtificial SequenceSynthetically generated peptide 270Gln Val
Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Pro1 5
10 15Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Val Arg Gln Ser Leu Ser Arg Gly Leu
Glu 35 40 45Trp Leu Gly Arg Thr
Tyr Tyr Arg Ser Lys Trp Tyr Asn Gly Tyr Ala 50 55
60Val Ser Val Arg Gly Arg Ile Thr Thr Asn Ala Asp Thr Ser
Arg Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr Gly
Tyr Ser Ser Ser Trp Val Val Asn Ser 100 105
110Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125271110PRTArtificial
SequenceSynthetically generated peptide 271Gln Pro Ala Leu Thr Gln Ser
Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile
Gly Ser Asn 20 25 30Tyr Val
Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly
Val Pro Gly Arg Phe Ser 50 55 60Gly
Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Ser Gly Leu Arg65
70 75 80Ser Lys Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 95Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr
Val Leu 100 105
110272258PRTArtificial SequenceSynthetically generated peptide 272Gln Val
Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5
10 15Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu
Glu 35 40 45Trp Leu Gly Arg Thr
Tyr Tyr Arg Thr Lys Trp Tyr Asn Glu Tyr Ala 50 55
60Ala Ser Val Lys Gly Arg Ala Thr Ile Asn Pro Asp Thr Ser
Lys Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Thr Asp Pro
Lys Gly Val Thr Thr Gln Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala
Ser Thr 115 120 125Gly Ile Leu Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130
135 140Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Pro
Ser Ala Ser Gly145 150 155
160Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn
165 170 175Ile Gly Arg Asn Tyr
Val Tyr Trp Tyr Gln Arg Leu Pro Gly Thr Ala 180
185 190Pro Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro
Ser Gly Ala Pro 195 200 205Ala Arg
Phe Ser Gly Ser Lys Ser Gly Thr Ser Thr Ser Leu Ala Ile 210
215 220Ser Gly Leu Arg Ser Glu Asp Glu Ala Glu Tyr
Phe Cys Ala Ala Trp225 230 235
240Asp Asp Ser Leu Ser Gly Trp Val Phe Gly Gly Gly Thr Gln Leu Thr
245 250 255Val
Leu273255PRTArtificial SequenceSynthetically generated peptide 273Gln Val
Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5
10 15Thr Leu Ser Leu Thr Cys Gly Ile
Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu
Glu 35 40 45Trp Leu Gly Arg Thr
Tyr Tyr Arg Ser Arg Trp Tyr Asn Asp Tyr Ala 50 55
60Ala Ser Val Lys Ser Arg Ile Thr Val Asn Ala Asp Thr Ser
Lys Asn65 70 75 80Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Ser Val
Arg Tyr Ser Ser Gly Trp Gly Phe Asp 100 105
110Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ile
Leu Gly 115 120 125Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130
135 140Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly
Thr Pro Gly Gln145 150 155
160Arg Ile Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn
165 170 175Tyr Val Tyr Trp Tyr
Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 180
185 190Val Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro
Asp Arg Phe Ser 195 200 205Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 210
215 220Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala
Trp Asp Asp Ser Leu225 230 235
240Ser Gly Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
245 250 255274254PRTArtificial
SequenceSynthetically generated peptide 274Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val
Ser Arg Asn 20 25 30Ser Ala
Ala Trp Asn Leu Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Thr Asp Tyr Ala 50 55 60Val
Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Arg Leu
Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Tyr Cys Ala Arg Ser Gly Gly Gly His Ala Ala
Gly Lys Phe Asp 100 105 110Ser
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Ile Leu Gly 115
120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser 130 135
140Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln145
150 155 160Arg Val Thr Ile
Pro Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Lys 165
170 175Tyr Val Tyr Trp Tyr Gln His Leu Pro Gly
Thr Ala Pro Lys Leu Leu 180 185
190Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
195 200 205Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu Arg 210 215
220Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser
Leu225 230 235 240Ser Ala
Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 245
250275248PRTArtificial SequenceSynthetically generated peptide
275Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Asp Asp Arg Ala Val
85 90 95Tyr Tyr Cys Arg Gly Arg
Leu Gly Gly Gly Met Asp Val Trp Gly Gln 100
105 110Gly Thr Thr Val Thr Val Ser Ser Gly Ile Leu Gly
Ser Gly Gly Gly 115 120 125Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val Leu 130
135 140Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly
Gln Arg Val Thr Ile145 150 155
160Ser Cys Ser Gly Ser Ser Ser Asn Ile Asn Tyr Val Tyr Trp Tyr Gln
165 170 175His Leu Pro Gly
Thr Ala Pro Lys Leu Leu Ile Tyr Thr Asn Asn Arg 180
185 190Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly
Ser Lys Ser Gly Thr 195 200 205Ser
Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp 210
215 220Tyr Phe Cys Ala Ala Trp Asp Asp Ser Leu
Ser Gly Trp Val Phe Gly225 230 235
240Gly Gly Thr Lys Val Thr Val Leu
245276252PRTArtificial SequenceSynthetically generated peptide 276Gln Val
Gln Leu Val Gln Ser Glu Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Gly Val
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Ser Tyr
Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Val Thr Thr Gly
Ile Thr Arg Tyr Phe Asp Leu Trp Gly 100 105
110Arg Gly Thr Leu Val Thr Val Ser Ser Gly Ile Leu Gly Ser
Gly Gly 115 120 125Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val 130
135 140Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly
Gln Ser Ile Thr145 150 155
160Ile Ser Cys Thr Gly Thr Asn Ser Asp Ile Gly Gly Tyr Asn Tyr Val
165 170 175Ser Trp Tyr Gln Gln
His Pro Gly Lys Ala Pro Lys Leu Met Ile Phe 180
185 190Glu Val Thr Asn Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser Ala Ser 195 200 205Lys Ser
Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Asp 210
215 220Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala
Gly Ser Asn Thr Pro225 230 235
240Ser Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu
245 250277250PRTArtificial SequenceSynthetically
generated peptide 277Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20
25 30Ser Ala Ala Trp Asn Trp Ile Arg
Gln Ser Pro Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala
50 55 60Val Ser Val Lys Gly Arg Ile Thr
Ile Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp
Thr Ala Met 85 90 95Tyr
Tyr Cys Val Arg Ser Gly Gly Gly Arg Val Asp Pro Trp Gly Gln
100 105 110Gly Thr Leu Val Thr Val Ser
Ser Gly Ile Leu Gly Ser Gly Gly Gly 115 120
125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val
Leu 130 135 140Thr Gln Ser Pro Ser Ala
Ser Gly Thr Pro Gly Gln Arg Val Thr Ile145 150
155 160Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser
Asn Tyr Val Tyr Trp 165 170
175Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Arg Asn
180 185 190His Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser Ala Ser Lys Ser 195 200
205Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu
Asp Glu 210 215 220Ala Asp Tyr Tyr Cys
Ala Ala Trp Asp Asp Ser Leu Ser Gly Tyr Val225 230
235 240Phe Gly Thr Gly Thr Lys Leu Thr Val Leu
245 250278255PRTArtificial
SequenceSynthetically generated peptide 278Gln Val Gln Leu Val Gln Ser
Glu Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asn Asp Tyr 20 25 30Ala Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys
Tyr Tyr Thr Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asp Ser Leu
Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Glu Ala Ser Ser Gly Trp Tyr Ile Asp Ser
Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Gly Ile 115
120 125Leu Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly 130 135
140Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser145
150 155 160Pro Gly Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser 165
170 175Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg 180 185
190Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
195 200 205Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg 210 215
220Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly
Ser225 230 235 240Ser Gln
Val Thr Phe Gly Gln Gly Thr Arg Leu Glu Val Lys Ser 245
250 255279262PRTArtificial
SequenceSynthetically generated peptide 279Arg Val Gln Leu Gln Gln Leu
Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Phe Gly Asp Ser Val
Ser Ser Asn 20 25 30Gly Ala
Ala Trp Ser Trp Ile Arg Gln Ser Leu Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Ala Tyr Tyr Arg Ser Lys
Trp Tyr Asn Asp Tyr Ala 50 55 60Val
Ser Val Arg Gly Arg Ile Thr Ile Asn Ala Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu
Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Tyr Cys Ala Arg Thr Gly Tyr Ser Ser Ser Arg
Val Val Ser Ser 100 105 110Gly
Tyr Trp Gly Gln Gly Thr Leu Val Ala Val Ser Ser Gly Ser Ala 115
120 125Ser Ala Pro Ile Gly Ile Leu Gly Ser
Gly Gly Gly Gly Ser Gly Gly 130 135
140Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Pro145
150 155 160Ser Ala Ser Gly
Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly 165
170 175Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val
Tyr Trp Tyr Gln Gln Leu 180 185
190Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys Ser Asn Arg Arg Pro
195 200 205Ser Gly Val Pro Gly Arg Phe
Ser Gly Ser Lys Ser Gly Thr Ala Ala 210 215
220Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr
Tyr225 230 235 240Cys Ala
Ala Trp Asp Asp Ser Leu Arg Gly Tyr Val Phe Gly Thr Gly
245 250 255Thr Lys Leu Thr Val Leu
260280262PRTArtificial SequenceSynthetically generated peptide 280Gln
Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Pro1
5 10 15Thr Leu Ser Leu Thr Cys Ala
Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Val Arg Gln Ser Leu Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Gly Tyr Ala 50 55
60Val Ser Val Arg Gly Arg Ile Thr Thr Asn Ala Asp Thr
Ser Arg Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Thr
Gly Tyr Ser Ser Ser Trp Val Val Asn Ser 100
105 110Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Ser Ala 115 120 125Ser Ala
Pro Thr Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly 130
135 140Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Ala
Leu Thr Gln Ser Pro145 150 155
160Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly
165 170 175Ser Ser Ser Asn
Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Leu 180
185 190Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Lys
Asn Asn Gln Arg Pro 195 200 205Ser
Gly Val Pro Gly Arg Phe Ser Gly Ser Lys Ser Gly Thr Ala Ala 210
215 220Ser Leu Ala Ile Ser Gly Leu Arg Ser Lys
Asp Glu Ala Asp Tyr Tyr225 230 235
240Cys Ala Ala Trp Asp Asp Ser Leu Arg Gly Tyr Val Phe Gly Thr
Gly 245 250 255Thr Lys Leu
Thr Val Leu 26028118PRTArtificial SequenceSynthetically
generated peptide 281Arg Thr Tyr Tyr Arg Thr Lys Trp Tyr Asn Glu Tyr Ala
Ala Ser Val1 5 10 15Lys
Gly2829PRTArtificial SequenceSynthetically generated peptide 282Asp Pro
Lys Gly Val Thr Thr Gln Tyr1 528313PRTArtificial
SequenceSynthetically generated peptide 283Ser Gly Ser Thr Ser Asn Ile
Gly Arg Asn Tyr Val Tyr1 5
1028412PRTArtificial SequenceSynthetically generated peptide 284Lys Leu
Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser1 5
1028511PRTArtificial SequenceSynthetically generated peptide 285Ala Ala
Trp Asp Asp Ser Leu Ser Gly Trp Val1 5
1028618PRTArtificial SequenceSynthetically generated peptide 286Arg Thr
Tyr Tyr Arg Ser Arg Trp Tyr Asn Asp Tyr Ala Ala Ser Val1 5
10 15Lys Ser28712PRTArtificial
SequenceSynthetically generated peptide 287Ser Val Arg Tyr Ser Ser Gly
Trp Gly Phe Asp Tyr1 5
1028813PRTArtificial SequenceSynthetically generated peptide 288Ser Gly
Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Tyr1 5
1028912PRTArtificial SequenceSynthetically generated peptide 289Lys
Leu Leu Val Tyr Arg Asn Asn Gln Arg Pro Ser1 5
1029012PRTArtificial SequenceSynthetically generated peptide 290Ala
Ala Trp Asp Asp Ser Leu Ser Gly Arg Trp Val1 5
102917PRTArtificial SequenceSynthetically generated peptide 291Arg
Asn Ser Ala Ala Trp Asn1 529218PRTArtificial
SequenceSynthetically generated peptide 292Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr Thr Asp Tyr Ala Val Ser Val1 5 10
15Lys Ser29312PRTArtificial SequenceSynthetically
generated peptide 293Ser Gly Gly Gly His Ala Ala Gly Lys Phe Asp Ser1
5 1029413PRTArtificial SequenceSynthetically
generated peptide 294Ser Gly Ser Ser Ser Asn Ile Gly Ser Lys Tyr Val Tyr1
5 1029511PRTArtificial
SequenceSynthetically generated peptide 295Ala Ala Trp Asp Asp Ser Leu
Ser Ala Trp Val1 5 102967PRTArtificial
SequenceSynthetically generated peptide 296Ser Asn Ser Ala Thr Trp Asn1
529710PRTArtificial SequenceSynthetically generated peptide
297Gly Gly Arg Leu Gly Gly Gly Met Asp Val1 5
1029811PRTArtificial SequenceSynthetically generated peptide 298Ser
Gly Ser Ser Ser Asn Ile Asn Tyr Val Tyr1 5
1029912PRTArtificial SequenceSynthetically generated peptide 299Lys Leu
Leu Ile Tyr Thr Asn Asn Arg Arg Pro Ser1 5
1030017PRTArtificial SequenceSynthetically generated peptide 300Val Ile
Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val Lys1 5
10 15Gly30112PRTArtificial
SequenceSynthetically generated peptide 301Arg Val Thr Thr Gly Ile Thr
Arg Tyr Phe Asp Leu1 5
1030214PRTArtificial SequenceSynthetically generated peptide 302Thr Gly
Thr Asn Ser Asp Ile Gly Gly Tyr Asn Tyr Val Ser1 5
1030312PRTArtificial SequenceSynthetically generated peptide
303Lys Leu Met Ile Phe Glu Val Thr Asn Arg Pro Ser1 5
1030411PRTArtificial SequenceSynthetically generated peptide
304Ser Ser Tyr Ala Gly Ser Asn Thr Pro Ser Val1 5
1030518PRTArtificial SequenceSynthetically generated peptide
305Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1
5 10 15Lys
Gly3068PRTArtificial SequenceSynthetically generated peptide 306Ser Gly
Gly Gly Arg Val Asp Pro1 530713PRTArtificial
SequenceSynthetically generated peptide 307Ser Gly Ser Arg Ser Asn Ile
Gly Ser Asn Tyr Val Tyr1 5
1030812PRTArtificial SequenceSynthetically generated peptide 308Lys Leu
Leu Ile Tyr Arg Asn His Gln Arg Pro Ser1 5
1030911PRTArtificial SequenceSynthetically generated peptide 309Ala Ala
Trp Asp Asp Ser Leu Ser Gly Tyr Val1 5
103105PRTArtificial SequenceSynthetically generated peptide 310Asp Tyr
Ala Met His1 531117PRTArtificial SequenceSynthetically
generated peptide 311Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Thr Asp
Ser Val Lys1 5 10
15Gly31210PRTArtificial SequenceSynthetically generated peptide 312Glu
Ala Ser Ser Gly Trp Tyr Ile Asp Ser1 5
103139PRTArtificial SequenceSynthetically generated peptide 313Gln Gln
Tyr Gly Ser Ser Gln Val Thr1 53147PRTArtificial
SequenceSynthetically generated peptide 314Ser Asn Gly Ala Ala Trp Ser1
531518PRTArtificial SequenceSynthetically generated peptide
315Arg Ala Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1
5 10 15Arg
Gly31613PRTArtificial SequenceSynthetically generated peptide 316Thr Gly
Tyr Ser Ser Ser Arg Val Val Ser Ser Gly Tyr1 5
1031712PRTArtificial SequenceSynthetically generated peptide 317Lys
Leu Leu Ile Tyr Lys Ser Asn Arg Arg Pro Ser1 5
1031818PRTArtificial SequenceSynthetically generated peptide 318Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Gly Tyr Ala Val Ser Val1
5 10 15Arg Gly31913PRTArtificial
SequenceSynthetically generated peptide 319Thr Gly Tyr Ser Ser Ser Trp
Val Val Asn Ser Asn Tyr1 5 10
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