Patent application title: CELL SENESCENCE MARKERS AS DIAGNOSTIC AND THERAPEUTIC TARGETS
Inventors:
Neil R. Cashman (Vancouver, CA)
Neil R. Cashman (Vancouver, CA)
IPC8 Class: AC07K1628FI
USPC Class:
424 91
Class name: Drug, bio-affecting and body treating compositions in vivo diagnosis or in vivo testing
Publication date: 2016-04-28
Patent application number: 20160115237
Abstract:
Provided are methods and agents for depleting senescent cells endogenous
to a subject, involving administering to the subject a binding agent that
is selectively toxic to senescent cells in an amount effective to reduce
the number of such cells, wherein the binding agent binds selectively to
a senescent cell surface protein having a misfolded conformation,
relative to said protein in a native conformation.Claims:
1. A method for depleting senescent cells endogenous to a subject,
comprising administering to the subject a binding agent that is
selectively toxic to senescent cells in an amount effective to reduce the
number of such cells, wherein the binding agent binds selectively to a
senescent cell surface protein having a misfolded conformation, relative
to said protein in a native conformation.
2. The method according to claim 1, wherein the binding agent binds selectively to a misfolded form of human PrP.
3. The method according to claim 2, wherein the binding agent binds a misfolded human PrP epitope comprising a YYR motif or a YML motif.
4. (canceled)
5. The method according to claim 2, wherein the binding agent binds a misfolded human PrP epitope comprising MDEYSNQNN (SEQ ID No.9).
6. The method according to claim 1, wherein the binding agent binds selectively to a misfolded form of human Fas receptor.
7. The method according to claim 6, wherein the binding agent binds a misfolded human Fas receptor epitope comprising LHHDGQFCH (SEQ ID No. 62) or NSTVCEH (SEQ ID No. 65).
8. (canceled)
9. The method according to claim 1, wherein the binding agent binds selectively to a misfolded form of a surface protein selected from the human forms of Fas ligand, CD44, EGF receptor, CD38, Notch-1, CD44, CD59, and TNF receptor.
10. The method according to claim 1, wherein the binding agent is an antibody or a binding fragment thereof.
11. The method according to claim 10, wherein the antibody is a misfolded human PrP antibody that binds selectively to an epitope having the sequence of any one of SEQ ID Nos. 1-14.
12. The method according to claim 11, wherein the misfolded human PrP antibody is an antibody produced by, or that binds to the same epitope as the antibody produced by the hybridoma cell line deposited with the International Depositary Authority of Canada under accession number 260210-01.
13. The method according to claim 11, wherein the misfolded human PrP antibody is an antibody comprising the following CDRs: For the heavy chain: TABLE-US-00016 (SEQ ID No. 41) CDR1 TYAMG (SEQ ID No. 42) CDR2 VITKSGNTYYASWAKG (SEQ ID No. 43) CDR3 YGIGVSYYDI
For the light chain: TABLE-US-00017 (SEQ ID No. 44) CDR1 QSSQSLYNKNWLS (SEQ ID No. 45) CDR2 KASTLES (SEQ ID No. 46) CDR3 QGEFSCSSADCTA.
14. The method according to claim 13, wherein the antibody is the antibody c-120.
15. The method according to claim 10, wherein the antibody is a misfolded human Fas receptor antibody that binds selectively to an epitope having the sequence of any one of SEQ ID Nos. 62-66.
16. The method according to claim 1, for the treatment of an age-related degenerative disorder.
17. The method according to claim 16, for the treatment of cancer, or emphysema, or aging of the skin, or sarcopenia.
18.-20. (canceled)
21. The method according to claim 1, wherein the binding agent is a conjugate comprising an antibody or binding fragment thereof, and a second agent that is a toxin or a detectable label.
22. (canceled)
23. A method for detecting senescent cells, comprising combining (1) a binding agent that binds selectively to a senescent cell surface protein having a misfolded conformation, and (2) a sample containing cells to be screened, and then determining the formation of a complex therebetween, the presence of such complex indicating that the sample contains a senescent cell.
24. The method according to claim 24, wherein the binding agent further comprises a detectable label.
25. The method according to claim 23, wherein the senescent state of the cell is confirmed by a second senescent cell detection method; optionally wherein the second senescent cell detection method is a test for B-galactosidase activity at pH6.
26. (canceled)
27. A method for imaging senescent cells in a subject, comprising administering to the subject an imaging agent comprising a detectable label and, conjugated thereto, a binding agent that binds selectively to a senescent cell surface protein having a misfolded conformation, allowing the imaging agent to bind to any such senescent cells, and detecting the location of the detectable label in said subject.
28. A diagnostic kit comprising (1) an antibody that binds selectively to a senescent cell surface protein having a misfolded conformation, and (2) written instructions for the use thereof to detect senescent cells in accordance with the method defined in claim 23.
Description:
RELATED APPLICATIONS
[0001] This is a Patent Cooperation Treaty Application which claims the benefit of priority of U.S. Provisional Patent Application No. 61/827,255, filed May 24, 2013 which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to markers of cell senescence, and to applications thereof as targets for medical diagnosis and therapeutic intervention. More particularly, the invention relates to surface proteins that manifest in senescent cells, and to agents such as antibodies that bind such surface proteins for use in diagnosing and treating related disease conditions.
BACKGROUND TO THE INVENTION
[0003] Senescence is generally defined as a cessation of cell replication that follows after a finite number of population doublings. Senescent cells remain metabolically active but do not divide and resist apoptosis for long periods. Cellular senescence is characterized by growth cycle arrest in the G1 phase, absence of S phase and lifespan control by multiple dominant genes.
[0004] Cellular senescence differs from quiescence and terminal differentiation. Senescent cells manifest morphological changes such as enlargement, flattening and increased granularity. They do not divide even if stimulated by mitogens. Replicative senescence is related to the progressive shortening of telomeres. Senescence is induced when certain chromosomal telomeres reach a critical length, and can be abrogated by the expression of telomerase which lengthens telomeres. Human fibroblasts, for instance, undergo replication indefinitely when transfected to express telomerase. Evidence suggests some relationship between replicative senescence and aging.
[0005] There are pathways to senescence other than replication. These are often referred to as stress-induced premature senescence (SIPS). Oxidative stress can shorten telomeres, and hyperoxia has been shown to induce senescence. Gamma irradiation of human fibroblasts in early to mid G1 phase causes senescence in a p53-dependent manner, and UV radiation also induces cellular senescence. Other agents that can induce cellular senescence include hydrogen peroxide, sodium butyrate, 5-azacytidine, and transfection with the Ras oncogene. Senescence in cancer cells can be induced by such chemotherapeutics as doxorubicin, cisplatin, and 5-bromodeoxyuridine, the latter being an agent that induces senescence in both normal and cancer cells.
[0006] The existence of cellular senescence has been demonstrated in vivo. Senescent fibroblasts were shown to exhibit staining for β-galactosidase activity at pH 6. These cells failed to incorporate tritiated thymidine and retained B-galactosidase activity after replating but did not divide. Quiescent fibroblasts did not show staining. Keratinocytes, umbilical vein endothelial cells and mammary epithelial cells all showed increased staining with increased population doublings. Immortalized cells and terminally differentiated keratinocytes did not show staining. Staining was performed on skin biopsies to test whether senescence is observed in vivo. An age-dependent pattern emerged in which an increased number of cells showed staining with increase donor age, in the dermis and epidermis. The existence of an increase in the number of senescent fibroblasts has been shown in the lungs of subjects with emphysema.
[0007] Cancer stem cells are immortal, meaning that they can replicate indefinitely without exhibiting senescence. Likely, the teleological purpose of senescence is to prevent cancer by limiting the number of cell divisions that can occur. Senescent cells can also promote tumorigenesis. Senescent stromal cells express tumour promoting as well as tumour suppressing factors that exert an effect on neighbouring epithelial cells, such as mitogenicity and anti-apoptosis.
[0008] In a dramatic demonstration, Baker et al report that a variety of age-related conditions were significantly improved in mice when senescent cells were removed (Nature, 2011, 479:232-236). Treated mice had substantially delayed lordokyphosis (a measure of sarcopenia) and cataracts, as well as increased muscle fiber diameter, reduced loss of adipose tissue, and improved skin tone. Some improvements were seen not only in younger mice, but also in older recipients in which age-related conditions were already established.
[0009] In summary, cellular senescence does occur in vivo and is a likely sequel to environmental insults. Its prevalence increases with age at least in some tissue compartments. Senescence confers functional changes on the cell which have been associated with various age-related diseases that include degenerative disorders such as Alzheimer's disease, cardiovascular disease, emphysema, sarcopenia, and tumorigenesis as well as conditions more cosmetic in nature such as signs of skin aging including wrinkling, sagging, discolouration, and the like. Senescent cells also develop a so-called senescence associated secretory phenotype (SASP) which enables cells to secrete a variety of growth factors, cytokines, and proteases that contribute to age-related tissue dysfunction as well as to tumour formation, and thus create a local environment that is toxic to neighbouring normal cells.
[0010] There is accordingly a need for agents that are capable of detecting senescent cells and for depleting senescent cells, thereby to diagnose and treat conditions associated with cell senescence.
SUMMARY OF THE INVENTION
[0011] It has now been discovered that senescent cells manifest a surface marker that can be targeted for therapeutic and diagnostic purposes. The marker is related to a surface protein that is native to the cell in its healthy state. Particularly, it has been found that certain native surface proteins adopt a misfolded conformation when the host cell is senescent. Accordingly, the present invention exploits, as target, a misfolded form of a senescent cell surface protein. The misfolded surface protein is unique in conformational terms relative to the native protein, and allows for the production and use of binding agents, such as antibodies, that bind selectively to the misfolded form of the protein.
[0012] In one of its aspects, the present invention provides a method for detecting senescent cells, in which a binding agent that binds selectively to a misfolded surface protein, i.e., the marker protein, is combined with a sample containing cells to be examined, and determining whether the cells are bound by the binding agent to form a complex, the presence of such complex indicating that the sample contains senescent cells.
[0013] The assay method can be performed using techniques such as flow cytometry, ELISA, Western blot and the like. The assay can be practised in vitro, and in vivo by way of imaging to reveal senescent cells endogenous to a subject.
[0014] In another of its aspects, the present invention provides a method useful to deplete senescent cells endogenous to a subject and/or to treat an age-related disease in a subject in need thereof, comprising administering to the subject a binding agent that is selectively toxic to senescent cells in an amount effective to reduce the number of such cells, wherein the binding agent binds selectively to a senescent cell surface protein having a misfolded conformation.
[0015] In embodiments, the binding agent is an antibody or fragment thereof that binds selectively to the marker protein. Suitable binding agents are agents that bind the marker protein at least two fold more efficiently than its native protein counterpart. Particularly suitable binding agents are antibodies, and fragments thereof that bind the marker protein with comparable efficiency. Also suitable are antibody conjugates in which the antibody or binding fragment is conjugated with a toxin useful to kill the targeted senescent cells.
[0016] In a further aspect, the present invention provides a method useful to deplete senescent cells endogenous to a subject in need thereof and/or to treat an age-related disease in a subject in need thereof, comprising administering to the subject an immunogen, such as in the form of a vaccine, that elicits an antibody that is selectively toxic to senescent cells, wherein the antibody binds selectively to a senescent cell surface protein having a misfolded conformation.
[0017] In one specific embodiment, the marker protein is a misfolded form of prion protein, PrP. In other embodiments, the marker protein is a misfolded form of a cell surface protein such as Fas receptor (FasR). In further embodiments, the marker protein is a misfolded form of CD38, Notch-1, CD44, CD59, Fas ligand, TNF receptor, or EGF receptor.
[0018] In embodiments, the present method exploits particularly an antibody that binds selectively to a misfolded form of human FasR, as well as misfolded FasR-binding antibody fragments and conjugates thereof, and pharmaceutical compositions and medical uses thereof. In addition, there is provided a method for producing the antibody, and immunogens/vaccines that elicit endogenous production of the antibody.
[0019] These and other aspects of the invention are now described in greater detail with reference to the accompanying drawings in which:
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows gating of live cells by flow cytometry based on forward scatter (FSC), side scatter (SSC), and viability dye exclusion (PerCP-Cy5.5).
[0021] FIG. 2 shows the binding of isotype control antibodies (mIgG and rIgG, mouse IgG and rabbit IgG, respectively), secondary antibody alone (2° Ab), the pan-PrP specific antibody 6D11, and the misfolding specific anti-PrP antibody AMF-1c-20 (DSE) to untreated and mitomycin c-treated senescent HUVEC cells. Analysis is by flow cytometry.
[0022] FIG. 3 shows gating of live cells by flow cytometry based on forward scatter (FSC), side scatter (SSC), and 7AAD viability dye exclusion (PerCP-Cy5.5).
[0023] FIG. 4 shows binding isotype control antibodies (mIgG and rIgG, mouse IgG and rabbit IgG, respectively), secondary antibody alone (2° anti-m and 2° anti-r), the pan-PrP specific antibody 6D11, and the misfolding specific anti-Fas antibodies AMF-3a-118 and AMF-3d-19 to untreated (A) and mitocmycin c-treated senescent (B) HUVEC cells. Analysis is by flow cytometry.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The invention is based on the discovery of a senescent cell marker that can be targeted for diagnostic and medical intervention. The marker is a cell surface protein that has adopted a misfolded conformation on cells that are senescent. The marker is thus related to a protein found in a natively folded state on the surface of a normal, healthy cell. When the cell has entered a senescent state, however, the marker protein is misfolded, and presents regions or domains that are not found or accessible in the natively folded protein. These unique regions or domains serve as epitopes for the raising of antibodies and selection of other binding agents that, in turn, bind uniquely and selectively to the misfolded protein.
[0025] The present methods are applied diagnostically to detect senescent cells present in any given sample, including biological fluids or tissues obtained from a subject, or to detect senescent cells in situ, i.e., senescent cells that are endogenous to a subject.
[0026] The present methods are also applied therapeutically to deplete senescent cells endogenous to a subject in need of treatment. Senescent cell "depletion" refers simply to a reduction in the number of viable senescent cells. Without wishing to be bound by theory, the result of eliminating or depleting senescent cells is to reduce the effect of these cells on their neighbouring environment, such as to reduce the so-called senescence-associated secretory phenotype (SASP) effect through which senescent cells create a toxic local environment that affects the health of neighbouring cells and promotes cell aging and associated medical conditions, including tumourigenesis.
[0027] The invention is thus based on the principle that misfolded proteins are present on the surface of senescent cells, and that senescent cells can therefore be identified and their deleterious effects controlled, reduced or eradicated by targeting the misfolded form of that surface protein. In one approach, senescent cells are screened to identify surface proteins that have adopted a conformation different from normal cells that present the protein in natively folded state. Once identified, the surface protein can be examined in silico using established algorithms to identify misfolding "hot spots" within its sequence. One such algorithm is described by Cashman et al in WO 2010/040209, incorporated herein by reference. As described in that publication, a wide variety of cell surface proteins have been examined and epitopes that are uniquely presented when that protein misfolds are identified. These epitopes are particularly useful targets for the methods of the present invention.
[0028] Senescent cell surface protein epitopes that are presented "uniquely" by the misfolded form of a protein, relative to the natively folded form of that protein, thus provide a basis for distinguishing between the two forms of the protein, and allow the misfolded protein to be targeted while substantially removing the natively folded protein as a complicating factor in therapeutic and diagnostic applications. In principle, an epitope is unique to a misfolded protein if an antibody or other ligand that binds that epitope shows significantly reduced binding to the natively folded form of that protein. In an embodiment, the binding difference is effective to yield a discernible test result or therapeutic result. The misfolded form of a protein is characterized, in the present context, as a form that presents an epitope not found in the natively folded protein. This results from the protein adopting a different conformation, owing for example to different environmental conditions (for example, oxidation of cysteine), defects in protein folding surveillance (for example, ineffective chaperone function), aberrant post-translational modifications (for example, defective maturation of glycosylation), abnormal interaction with another protein (for example, PrPC/PrPSc interaction), or from genetic mutation. As a result of misfolding, the protein can adopt an unusual and undesirable state, with a different immunogenicity relative to its normal state.
[0029] Accordingly, an aspect of the invention includes a method to deplete senescent cells endogenous to a subject, comprising administering to the subject a binding agent that is selectively toxic to senescent cells in an amount effective to reduce the number of such cells, wherein the binding agent binds selectively to a senescent cell surface protein having a misfolded conformation.
[0030] The term "selectively toxic to senescent cells" means that the binding agent is at least 2 times more toxic to a senescent cell expressing the misfolded protein, compared to a cell such as a normal not expressing the misfolded protein, such as 3 times greater, 5 times greater or at least one order of magnitude greater (e.g., at least 2, 3, 4 or 5 orders of magnitude greater). For instance, the cell toxicity of an antibody that binds misfolded PrP on a senescent cell is preferably at least twice its toxicity for natively folded PrP on normal tissue.
[0031] Another aspect includes a method to treat an age-related disease in a subject in need thereof, comprising administering to the subject a binding agent that binds selectively to a senescent cell surface protein having a misfolded conformation.
[0032] The binding agent binds selectively to an epitope exposed in a misfolded conformation of the cell surface protein present on the senescent cell. The term "epitope" refers a region of a protein that is recognized by a B cell or T-cell receptor, or an antibody or a binding fragment thereof and is represented herein by linear peptides having amino acid sequences that are found in the parent protein in which the epitope resides. The epitope will usually comprise at least 3 contiguous amino acid residues, representing the minimal domain necessary for antibody binding. However, the epitope can include a larger number of contiguous residues, such as 5 residues, 7 residues which typically is the length required for minimal immunogenicity within common antibody production hosts, 10 residues, 15 residues, 20 residues or more. The criticality lies in providing an epitope that harbours an antibody-binding domain. Thus, and with reference to Tables 1, 2 and 3, infra, it is to be appreciated that the peptides specifically listed, in addition to serving per se as epitopes, may further comprise additional amino acid residues at one or both flanks thereof, particularly for those peptides that consist of fewer than 7 residues. The additional residues can be those normally associated with the peptide in the context of the parent protein. Conversely, it should be appreciated that truncated forms of the peptides can also serve as epitopes, especially when the peptide consists of more than 7 residues. The extent of truncation can vary depending on the actual length of a given peptide. Any contiguous 7 residues within a listed peptide can be expected to be immunogenic per se, and any 3, 4, 5, 6, or 7 contiguous residues or more can be expected to be useful as an epitope to which antibodies can bind.
[0033] The senescent cell marker can be any surface protein that is misfolded on the surface of a senescent cell, but is otherwise natively folded on a normal cell counterpart. Of course, the variety of surface proteins that are candidates as senescent cell markers are numerous. The determination that a given surface protein is a senescent cell marker can be made by comparing the conformations of that protein on a normal cell vs. a senescent cell of the same type. In embodiments, the surface protein is selected from prion protein (PrP), FasR, Fas ligand, CD44, EGF receptor, CD38, Notch-1, CD44, CD59, or TNF receptor, and the like.
Prion Protein, PrP, as Target
[0034] In embodiments of the present invention, the senescent cell marker is a misfolded form of prion protein, or PrP. In mature form, the human prion protein (hPrPC) is a 230 residue protein having the sequence set out in UniProtKB/SwissProt designation P04156. This protein is known to adopt a misfolded conformation when associated with neurodegenerative diseases including CJD, and when presented on the surface of certain cancer cells, including particularly ovarian cancer cells. Accordingly, various antibodies specific for the misfolded forms of PrP have been developed and are described in the literature. These antibodies and vaccines that produce them are useful in the present invention.
[0035] More particularly, the present invention exploits agents that bind uniquely to misfolded PrP, and particularly, in embodiments of the present invention, to an epitope comprised by any one of the following peptides:
TABLE-US-00001 TABLE 1 SEQ Protein Target (abbr) Residue ID UniProtKB/SwissProt # span Sequence No. Human prion protein 125-130 LGGYML 1 (hPrP) P04156 126-132 GGYMLGS 2 residues 1-230 140-147 HFGSDYED 3 143-147 SDYED 4 148-155 RYYRENMH 5 151-155 RENMH 6 160-166 QVYYRPM 7 165-174 PMDEYSNQNN 8 166-174 MDEYSNQNN 9 185-197 KQHTVTTTTKGEN 10 70-80 ARDCTVNGDEP 11 105-111 RLCDEGH 12 136-142 NSTVCEH 13 167-189 EEPSRSNLGWLCL 14
[0036] In a particular embodiment, the binding agent binds selectively to an epitope that comprises a YYR motif. As shown in Table 1, the YYR motif lies within PrP residues 160-166 (SEQ ID No.7) and within PrP residues 148-155 (SEQ ID No.5). These peptides and the epitopes therein are accordingly useful targets for the binding agent. It will be appreciated that variations of these specific peptides that also comprise YYR and are based also on the PrP protein sequence are included as useful targets for the binding agent. Such peptides will typically include at least 5 contiguous residues, for instance, RYYRE (SEQ ID No.15), RYYREN (SEQ ID No.16), RYYRENM (SEQ ID No.17), DRYYRENMH (SEQ ID No.18), DRYYRENM (SEQ ID No. 19), and VYYRPM (SEQ ID No.20), QVYYRP (SEQ ID No.21), QVYYR (SEQ ID No.22), QVYYRPMD (SEQ ID No.23), and the like.
[0037] In another embodiment, the binding agent is one that binds selectively to a misfolded PrP epitope that comprises a YML motif. This particular motif lies within PrP beta strand 1. As shown in Table 1 above, the YML motif is common within PrP peptides, LGGYML and GGYMLGS (SEQ ID Nos. 1 and 2, respectively). These peptides are accordingly also useful epitope targets for the binding agent. It will be appreciated that variations of these specific peptides that also comprise YML and are based also on parent protein sequence are included as useful epitope targets in embodiments of the present invention. Such peptides will typically include at least 3, 4 or more usually at least 5 contiguous residues, for instance, GGYMLG (SEQ ID NO: 25), GYMLGS (SEQ ID NO: 26), GGYML (SEQ ID NO: 27), YMLGS (SEQ ID NO: 28), GYML (SEQ ID NO: 29), YMLG (SEQ ID NO: 30) LGGYML (SEQ ID No. 31), LGGYMLG (SEQ ID No. 32) and YML (SEQ ID NO: 33).
[0038] In a further embodiment, the binding agent binds selectively to a misfolded PrP epitope that comprises a MDEYSNQNN motif. This particular motif, located between β-sheet 2 and α-helix 1, is known also as the rigid loop, and lies within PrP residues 167-174, as shown in Table 1. It will be appreciated that variations of these specific peptides that also comprise at least about 5 contiguous residues within the rigid loop sequence and are based also on parent protein sequence are included as useful epitope targets in embodiments of the present invention. Such peptides will typically include at least 5 contiguous residues, for instance, PMDEYSNQNN (SEQ ID No.34), DEYSNQNN (SEQ ID No.35), and MDEYSNQ (SEQ ID No.36).
[0039] Agents that bind to these epitopes will bind selectively to misfolded PrP, relative to natively folded PrP, and will thus bind selectively to senescent cells in which surface PrP is misfolded, relative to normal cells.
[0040] Agents that bind to these epitopes can be identified using routine screening methods in which a panel of putative ligands is combined with a peptide presenting the epitope, and binding events are then analyzed to identify peptide binding partners. Ligands that also bind to PrP in its wild type, natively folded state can be dismissed, so that only the ligands or binding partners that bind uniquely to the epitope targets are selected.
[0041] It will be appreciated that the binding agents useful herein can take the form of small molecules, peptides, peptidomimetics, peptide or nucleic acid aptamers, and the like. In particular embodiments, the binding agent is an antibody or an epitope-binding fragment thereof. In other embodiments, the binding agent is induced by the administration of an immunogen that elicits production within the recipient of antibodies that bind the senescent cell marker.
[0042] In embodiments, the binding agent is an antibody that binds selectively to PrP in a misfolded form presented by senescent cells. In particular embodiments, the antibody is one that binds selectively to a peptide listed in Table 1.
[0043] In more specific embodiments, the antibody is one that binds a peptide incorporating the YML motif of PrP. Particularly, the antibody is the antibody designated 1A1, as described in more detail in WO 2010/099612 incorporated herein by reference. This antibody displays selective binding to the prion protein YML motif. Its hybridoma source was deposited under terms of the Budapest Treat at the International Depositary Authority of Canada (IDAC) under accession number 260210-01.
[0044] In other specific embodiments, the antibody is one that binds a peptide incorporating the YYR motif of PrP. Particularly, the antibody is one that is raised in the manner described in WO 03/000853 (and see U.S. Pat. No. 7,041,807) incorporated herein by reference, and as reproduced in Example 1 herein for convenience.
[0045] In another specific embodiment, the antibody is one that binds the rigid loop of PrP. Particularly, the antibody is the antibody designated c-120, as described in co-pending WO2013/185215 published on 2013 Dec. 19, and incorporated herein by reference. This antibody is defined by heavy and light chains having the sequences set out in SEQ ID No.38 and SEQ ID No. 37, respectively.
[0046] The variable regions of the antibody have the sequences set out in SEQ ID No.39 for the light chain variable region, and in SEQ ID No.40 for the heavy chain variable region.
[0047] The sequences of the important complementarity determining regions of the c-120 antibody have the following sequences:
[0048] For the heavy chain:
TABLE-US-00002 (SEQ ID No. 41) CDR1 TYAMG (SEQ ID No. 42) CDR2 VITKSGNTYYASWAKG (SEQ ID No. 43) CDR3 YGIGVSYYDI
[0049] For the light chain:
TABLE-US-00003 (SEQ ID No. 44) CDR1 QSSQSLYNKNWLS (SEQ ID No. 45) CDR2 KASTLES (SEQ ID No. 46) CDR3 QGEFSCSSADCTA
[0050] It will be appreciated that the binding agent thus can be an antibody or a fragment thereof that binds selectively to the rigid loop of PrP and incorporates at least the CDR sequences provided above.
[0051] Cell surface proteins other than PrP are also misfolded when the host cell is in a state of senescence. Like PrP, the conformational alteration results in the presentation of new epitopes that are unique to the misfolded protein, relative to its native counterpart. These epitopes are useful targets for senescent cell detection and depletion. More particularly, the present inventors have applied an algorithm that predicts misfolding "hot spots" within surface proteins. That algorithm is described by Cashman et al in WO 2010/040209, and was used to identify the misfolding specific epitopes within the target surface proteins shown below.
TABLE-US-00004 TABLE 2 Protein Target (abbr) SEQ UniProtKB/SwissProt Residue ID # span Sequence No. Human CD44 P16070 39-45 NGRYSIS 47 residues 21-742 83-87 EGHVV 48 108-116 TSNTSNYDT 49 149-159 NRDGTRYVQKG 50 99-116 ANNTFVYILTSNTSNYDT 51 Human tumour 21-31 IHPQNNSICCT 52 necrosis 41-51 NDCPGPGQDTD 53 factor receptor 106-112 YWSENLF 54 (hTNFR)P19438 126-134 HLSCQEKQN 55 139-151 CTCHAGFFLRENECV 56 Human notch protein 423-429 CEHAGKC 57 (hNOTCH1)P46531 479-487 MPGYEGVHC 58 residues 1-2550 499-505 CLHNGRC 59 508-512 KINEF 60 493-517 ECASSPCLHNGRCLDKIN 61 EFQCECP Human Fas receptor 52-60 LHHDGQFCH 62 P25445 residues 26- 70-80 ARDCTVNGDEP 63 335 105-111 RLCDEGH 64 136-142 NSTVCEH 65 167-189 EEGSRSNLGWLCL 66 Human epidermal 11-25 SNKLTQLFTFEDHFL 67 growth factor 46-56 VQRNYDLSFLK 68 receptor 83-95 IRGNMYYENSYAL 69 (hEGFR)P00533 97-107 VLSNYDANKTG 70 residues 25-1210 143-155 IVSSDFLSNMSMD 71 148-169 FLSNMSMDFQNHLGS 72 176-181 WGAGEE 73 241-259 PPLMLYNPTTYQMDVNPE 74 257-266 PEGKYSFGAT 75 273-282 RNYVVTDHGS 76 288-301 GADSYEMEEDGVRK 77 314-328 NGIGIGEFKDSLSIN 78 328-337 ATNIKHFKN 79 348-363 LPVAFRGDSFTHTPPL 80 465-474 KIISNRGENS 81 Human CD38 70-84 YTEIHPEMRHVDCQS 82 P28907 1-300 161-169 GEFATSKIN 83 176-182 WRKDCSN 84 210-220 GSRSKIFDKDS 85 243-253 IHGGREDSRDL 86
[0052] It has been determined further that the cell surface protein human FasR (Fas receptor) is also misfolded when present on senescent cells. Fas receptor (FasR) is known also as human tumour necrosis factor superfamily member 6 receptor (hTNFRSM6), and as CD95, and is implicated in cancer. It is a death receptor on the surface of cells that leads to caspase-mediated programmed cell death (apoptosis). More particularly, the present invention provides and exploits agents that bind uniquely to misfolded FasR, and particularly, in embodiments of the present invention, to an epitope comprised by any one of the peptides shown in Table 3:
TABLE-US-00005 TABLE 3 Human FasR 52-60 LHHDGQFCH 62 (hTNFRSM6) 70-80 ARDCTVNGDEP 63 P25445 residues 26- 105-111 RLCDEGH 64 335 136-142 NSTVCEH 65 167-189 EEPSRSNLGWLCL 66
[0053] In a particular embodiment, the binding agent binds selectively to a FasR epitope that comprises an LHHDGQFCH sequence (SEQ ID No.62). In an alternative embodiment, the binding agent binds selectively to a FasR epitope that comprises an NSTVCEH sequence (SEQ ID No.65).
[0054] Also provided herein as binding agents that bind selectively to misfolded FasR, are antibodies that bind to one of the two peptide sequences just recited. In particular, and in one of its aspects, the present invention provides an antibody designated AMF 3a-118, the antibody having been raised against the LHHDGQFCH in the manner exemplified herein. The heavy chain of the 3a-118 antibody has SEQ ID No. 89. The light chain of the 3a-118 antibody has SEQ ID No. 91. The heavy chain variable region of the 3a-118 antibody has SEQ ID No. 90. The light chain variable region of the 3a-118 antibody has SEQ ID No. 92. The misfolded FasR binding site presented by this antibody comprises the following CDRs:
[0055] For the heavy chain
TABLE-US-00006 (SEQ ID No. 93) CDR1 DSRVS (SEQ ID No. 94) CDR2 IVGIGWNIYHANWAKG (SEQ ID No. 95) CDR3 GLGGGTVI
[0056] For the light chain
TABLE-US-00007 (SEQ ID No. 96) CDR1 QSSESVYKNNYLS (SEQ ID No. 97) CDR2 EASKLAS (SEQ ID No. 98) CDR3 LGEFSCYSGDCGT
[0057] In addition, the present invention provides an antibody designated AMF 3d-19, the antibody having been raised against the NSTVCEH sequence in the manner exemplified herein. The heavy chain of the 3d-19 antibody has SEQ ID No. 103. The light chain of the 3d-19 antibody has SEQ ID No. 105. The heavy chain variable region of the 3d-19 antibody has SEQ ID No. 104. The light chain variable region of the 3d-19 antibody has SEQ ID No. 106. The misfolded FasR binding site presented by this antibody comprises the following CDRs:
[0058] For the heavy chain
TABLE-US-00008 (SEQ ID No. 107) CDR1 RNAIN (SEQ ID No. 108) CDR2 IIGSSGVTYYASWAKG (SEQ ID No. 109) CDR3 NLYTGGSNDNL
[0059] For the light chain
TABLE-US-00009 (SEQ ID No. 110) CDR1 QASKSVYNNVQLS (SEQ ID No. 111) CDR2 YASTLAS (SEQ ID No. 112) CDR3 AGGYSSSSDNA
[0060] It will thus be appreciated that the binding agent useful in the present methods can be any antibody or a fragment thereof that binds selectively to a protein that is misfolded on the surface of a senescent cell, by binding to a particular epitope that is unique to that misfolded form relative to the natively folded protein.
[0061] The antibodies that bind selectively to the misfolding specific epitopes may be either polyclonal or monoclonal, of the IgG or IgM class, and may be derived from any mammal, particularly goats, rabbits or mice, or by recombinant methods. More generally, it will be appreciated that antibodies useful in the present invention include the various intact forms including polyclonal antibodies, monoclonal antibodies, and recombinant antibodies including chimeric antibodies, humanized antibodies as well as fully human antibodies and bispecific or multispecific antibodies.
[0062] In an embodiment, the antibody comprises a human constant region.
[0063] The chimeric antibodies comprise a portion of the heavy and/or light chain that is homologous with corresponding sequences in antibodies derived from a particular species, or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is homologous with corresponding sequences derived from another species or belonging to a different antibody class. Humanized antibodies are chimeric antibodies that comprise minimal sequence derived from non-human antibody, usually incorporating CDRs from a non-human antibody into a human antibody framework, which may further be altered to incorporate non-human residues that restore and enhance antigen binding. The "fully" human antibodies can be produced in a non-human host using various techniques that are now established, including through the use of phage display libraries, and particularly by introducing human immunoglobulin loci into transgenic animals such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibodies are produced which closely resemble that seen in humans in most respects, including gene rearrangement, assembly and antibody repertoire.
[0064] Using phage display antibodies are displayed on the surface of phage by for example fusing the coding sequence of antibody variable regions to the phage minor coat protein pill. Antibodies can be selected using the phage displayed antibody libraries (including synthetic antibody libraries wherein synthetic diversity is introduced at solvent-exposed positions within the heavy chain complementarity-determining regions) by a series of cycles of selection on antigen. Antibody genes can be cloned simultaneously with selection and further engineered for example by increasing their affinity or modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold e.g. making a recombinant human antibody).
[0065] In an embodiment, the antibody is a recombinant or synthetic antibody, for example the human antibody is a recombinant or synthetic human antibody.
[0066] The antibodies may be of any useful class, including IgA, IgD, IgE, IgG and IgM, and isotypes including IgG1, IgG2, IgG3, and IgG4. The constant region (Fc) of the antibodies can also be engineered or conjugated to provide altered effector function, thereby to enhance antibody dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) activity.
[0067] For therapeutic use, in embodiments, the antibody desirably is itself toxic to the senescent cells presenting the target epitope. To this end, the antibody desirably incorporates an Fc region having effector function, such as ADCC activity and/or CDC activity. In the alternative, and in accordance with other embodiments, the antibody or binding fragment is conjugated with a cytotoxin that is toxic to the senescent cells targeted by the antibody or binding fragment.
[0068] The antibodies otherwise can have all of the attributes common to intact antibodies. In embodiments, the present antibodies are of the IgG1 isotype, but they may also be IgG2 or IgG4. Moreover, the isotype of the antibody, as dictated by the constant region, can be manipulated to alter or eliminate the effector function of the resulting antibody. That is, the constant region of the present antibodies is either wild type human antibody constant region, or a variant thereof that incorporates amino acid modifications, i.e., amino acid additions, substitutions or deletions that alter the effector function of the constant region, such as to enhance serum half-life, reduce or enhance complement fixation, reduce or enhance antigen-dependent cellular cytotoxicity and improve antibody stability. The number of amino acid modifications in the constant region is usually not more than 20, such as 1-10 e.g., 1-5 modifications, including conservative amino acid substitutions.
[0069] In embodiments, the half-life of the antibody is improved by incorporating one or more amino acid modifications, usually in the form of amino acid substitutions, for instance at residue 252, e.g., to introduce Thr, at residue 254, e.g., to introduce Ser, and/or at residue 256 e.g., to introduce Phe. Still other modifications can be made to improve half-life, such as by altering the CH1 or CL region to introduce a salvage receptor motif, such as that found in the two loops of a CH2 domain of an Fc region of an IgG. Such alterations are described for instance in U.S. Pat. No. 5,869,046 and U.S. Pat. No. 6,121,022.
[0070] Altered C1 q binding, or reduced complement dependent cytotoxicity, can be introduced by altering constant region amino acids at locations 329, 331 and 322, as described in U.S. Pat. No. 6,194,551. The ability of the antibody to fix complement can further be altered by introducing substitutions at positions 231 and 239 of the constant region, as described in WO94/029351.
[0071] Framework modifications can also be made to reduce immunogenicity of the antibody or to reduce or remove T cell epitopes that reside therein, as described for instance by Carr et al in US2003/0153043.
[0072] Antibodies can also be altered in the variable region to eliminate one or more glycosylation sites, and/or to improve physical stability of the antibody. For example, in one embodiment, the physical stability of the antibody is improved by substituting the serine at position 228 of the variable region with a proline residue (i.e., the antibody has a variable region comprising a S228P mutation). The S228P alteration significantly stabilizes the antibody structure against the formation of intrachain disulfide bonds. In another embodiment, the variable region is altered to eliminate one or more glycosylation sites resident in the variable region. More particularly, it is desirable in the sequence of the present antibodies to eliminate sites prone to glycosylation. This is achieved by altering the occurrence of one or more N-X-(S/T) sequences that occur in the parent variable region (where X is any amino acid residue), particularly by substituting the N residue and/or the S or T residue.
[0073] Antibodies can be engineered to include a variety of constant regions. In one embodiment, the antibody comprises a constant region the sequence of which corresponds to the constant region of an antibody of human origin, such as a human IgG1 constant region. In a particular embodiment, the constant region is inert for effector function (e.g., essentially devoid of effector function). In a specific embodiment the constant region is a human IgG4 constant region.
[0074] In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fcγ receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072. Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcγRIII. Additionally, the following combination mutants were shown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.
[0075] In addition, the antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See, e.g., EP 154316 and EP 401384.
[0076] The antibodies and binding fragments are useful both for diagnostic purposes, including in vivo imaging to identify endogenous sites of senescent cells in vivo, and for sample testing to detect senescent cells in vitro. The antibodies and binding fragments are also useful for therapeutic purposes to treat diseases in which senescent cells are implicated.
[0077] "Selective-binding" agents are agents that bind the target epitope, and bind proteins that present the target epitope in a solvent-accessible orientation, with an affinity that is at least 2 times greater than the affinity with which they bind a different, unrelated epitope, such as 3 times greater, 5 times greater or at least one order of magnitude greater (e.g., at least 2, 3, 4 or 5 orders of magnitude greater For instance, the binding affinity of an antibody that binds misfolded PrP on a senescent cell is preferably at least twice its binding affinity for natively folded PrP on normal tissue. Relative binding affinities can be determined, and the antibody so selected, on the basis of assays and techniques that generally are well established in the art for this purpose.
[0078] In an embodiment, the antibody affinity has an EC50 which is at least 10-6 M, at least 10-7 M, at least 10-8 M, or at least 10-9 M.
[0079] All antibodies were then tested by ELISA for binding to denatured full-length recombinant PrP. One antibody exhibited a titer for denatured PrP that was similar to the anti-peptide affinities, in the 10-10 M range. Thus, the preferred antibody exhibits preferably an EC50 by this test that is at least better than 10-9M.
[0080] Antibodies that bind selectively to the epitope unique to a misfolded form of a protein can be produced by techniques including immunization, or by alternative approaches such as by the application of phage display and other systems that use high throughput to identify complementarity determining regions (CDR) or other sequences that bind to the target epitope. It is not essential that the resulting antibody has been first raised in vivo. More particularly, to produce suitable antibodies, amino acid sequences that constitute epitopes can be useful per se to raise antibodies that bind specifically to them, provided they are endowed per se with the immunogenicity required to raise antibody in the selected antibody production host. For those epitopes that lack such immunogenicity, it is desirable to provide an immunogen that contains the epitope sequence.
[0081] As used herein, "immunogen" refers to an immunogenic form of a peptide or other molecule that comprises the epitope, and is represented by the peptide itself when immunogenic per se, or is represented by the peptide in combination with an immunogenicity-enhancing agent. Any of the established agents can be used for this purpose. These agents typically include carrier proteins that can be coupled to the epitope either directly, such as through an amide bond, or indirectly through a chemical linker such as carbodiimide, a cysteine, or any peptide spacer sequence such as a glycine or glycine-serine sequence including Gly4-S. For example, an isolated peptide comprising a given epitope can be conjugated to MAP antigen, OVA antigen, or keyhole limpet hemocyanin (KLH). Its large size makes it very immunogenic, and the large number of lysine residues available for conjugation makes KLH very useful to attach to a polypeptide. The immunogen may further comprise a linker effective to couple the peptide tandemly to another copy of the same or a different peptide corresponding to the same or a different epitope. In another embodiment, the peptides may comprise additional amino acids that enhance the immunogenicity or solubility of the peptide. In one embodiment, the additional amino acids number from 1 to about 10, preferably 1 to 8, more preferably 1 to 5. Importantly the additional residues do not materially affect the conformation of the peptide.
[0082] Thus, for antibody production, epitopes that are not themselves immunogenic and do not constitute an immunogen can be rendered so, and provided as an immunogen, by incorporating immune enhancing agents that are either conjugated therewith or coupled covalently.
[0083] A composition comprising the immunogen can be prepared for purposes of producing antibodies in a selected host by combining the immunogen with an appropriate vehicle. Such vehicles include Freund's complete adjuvant or other adjuvant or a suitable saline or phosphate buffered saline solution (0.05-1.0%).
[0084] Antibodies are then prepared to react against these epitopes when they are in an unstructured state. As noted, each peptide may be conjugated to a carrier protein like KLH to form an immunogen that is injected, optionally in combination with an adjuvant such as Freund's complete adjuvant, into a mammalian production host like a mouse, rat, rabbit, sheep or goat to provoke an immune response that generates antibodies against the peptide. Standard immunization protocols can be used, and the antibodies can be recovered from blood by enrichment against the immunizing agent, as exemplified herein.
[0085] The antibodies useful herein are desirably "isolated" antibodies, which refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds misfolded PrP is substantially free of antibodies that specifically bind antigens other than PrP proteins). An isolated antibody that specifically binds a misfolded human PrP protein may, however, have cross-reactivity to other antigens, such as misfolded PrP proteins from other species. Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals. An isolated antibody also can be substantially free of other proteins of human origin. In embodiments, the isolated antibody is an exogenous antibody as distinct from an antibody endogenous to an intended recipient.
[0086] Thus, in embodiments, the antibody is an intact antibody comprising features common to all natural antibodies, e.g., a heavy chain and a light chain, each chain having a constant region and a variable region, each variable region comprising framework regions (FRs) and complementarity determining regions (CDRs). In the alternative, the antibody is provided as a target-binding fragment, such as a monovalent fragment, Fab, or a bivalent antibody fragment comprising both "arms" of an intact antibody, joined through a linker that can be represented by the hinge region of the antibody or any equivalent. Such fragments include F(ab)2 fragments and any other fragment that retains preference for binding to misfolded PrP. In particular embodiments, the antibody fragment is a F(ab')2 fragment, generated for instance by papain-based digestion of the parent antibody using standard procedures for digestion and subsequent fragment isolation. In the alternative, the fragment can be a so-called single chain Fv (scFv), consisting of the variable light and variable heavy antibody domains joined by an amino acid linker, or a bivalent form of a so-called diabody prepared using a 5 amino acid linker such as SGGGG (SEQ ID NO: 117) between the light and heavy chain variable domains and a C-terminal cysteine modification to GGC to give a final diabody product as VL-SGGG-VH-GGC. Still other bivalent fragments can be prepared by coupling the light and heavy chain variable domains through thioether linkages such as bis-maleimidomethyl ether (BMME), N,N'-p-phenylene dimaleimide (PDM and N,N'-bismaleimidohexane BMH), to stabilize the F(ab')2 fragments.
[0087] Of course, for antibodies having known protein or gene sequences, the antibody can be produced suitably by recombinant DNA means. For production, there is provided a DNA molecule that encodes the heavy chain of the present antibody, and a DNA molecule that encodes the light chain thereof. The DNA further encodes any suitable signal peptide suitable for expression of a secretable chain precursor that enables proper externalization with folding and disulfide formation to elaborate the desired antibody as a secreted, dimerized and processed protein.
[0088] To this end, the present invention provides, in one aspect, polynucleotides that encode the heavy and light chains of the FasR antibodies herein described. In one embodiment, there is provided a polynucleotide comprising a sequence that encodes the light chain variable region of the FasR antibody AMF 3a-118, as set out in SEQ ID No.102. Also provided, in another embodiment, is a polynucleotide comprising a sequence that encodes the heavy chain variable region of the FasR antibody AMF 3a-118, as set out in SEQ ID No.100.
[0089] In more specific embodiments, the present invention provides a polynucleotide that encodes the entire light chain (SEQ ID No. 101) and a polynucleotide that encodes the entire heavy chain (SEQ ID No. 99) of misfolded FasR antibody AMF 3a-118 antibody.
[0090] The present invention also provides, in another aspect, polynucleotides that encode the light chain variable region of the FasR antibody AMF 3d-19, as set out in SEQ ID No. 116, and a polynucleotide comprising a sequence that encodes the heavy chain variable region of the FasR antibody AMF 3d-19, as set out in SEQ ID No. 114.
[0091] In more specific embodiments, the present invention provides a polynucleotide that encodes the entire light chain (SEQ ID No. 115) and a polynucleotide that encodes the entire heavy chain (SEQ ID No. 113) of the misfolded FasR antibody AMF 3d-19
[0092] It will be appreciated that polynucleotide equivalents also can be used, in which synonymous codons are replaced within the sequences provided, to produce the present antibodies. In an embodiment, the nucleic acid is a cDNA. In another embodiment, the nucleic acid is a codon optimized cDNA.
[0093] In embodiments, there are also provided vectors that comprise polynucleotides that encode the heavy chain or the variable region thereof and that encode the light chain or the variable region thereof. To express the antibodies, the polynucleotides are incorporated operably within expression vectors, i.e. operatively linked to transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, cosmids, and the like. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy gene can be inserted into separate vectors. In a preferred embodiment, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
[0094] A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed, as described above. In such vectors, splicing usually occurs between the splice donor site in the inserted J region, and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
[0095] Polynucleotides encoding the heavy chain and/or the light chain, and vectors comprising these can be used for transformation of a suitable mammalian host cell. Methods for introduction of heterologous polynucleotides into mammalian calls include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, polynucleotides may be introduced into mammalian cells by viral vectors.
[0096] Mammalian cell lines useful as hosts for expression of the antibody-encoding polynucleotides include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells, and a number of other cell lines. In a specific embodiment, the polynucleotides are expressed in a HEK293 host. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as S19 cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be, recovered from the culture medium using standard protein purification methods.
[0097] The antibodies of the invention can be obtained as human monoclonal antibodies. Such human monoclonal antibodies can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as the HuMAb Mouse® and KM Mouse®, respectively, and are collectively referred to herein as "human Ig mice."
[0098] The HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (see e.g., Lonberg et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGκ monoclonal antibodies (Lonberg et al. (1994), supra; reviewed in Lonberg (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding and Lonberg (1995) Ann. N.Y. Acad. Sci. 764:536-546). Preparation and use of the HuMAb Mouse®, and the genomic modifications carried by such mice, is further described in Taylor et al. (1992) Nucleic Acids Research 20:6287-6295; Chen et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor et al. (1994) International Immunology 6: 579-591; and Fishwild et al. (1996) Nature Biotechnology 14: 845-851, the contents of all of which are hereby specifically incorporated by reference in their entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and 5,545,807; PCT Publication Nos. WO 92/03918; WO 93/12227; WO 94/25585; WO 97/13852; WO 98/24884; WO 99/45962 and WO 01/14424, the contents of which are incorporated herein by reference in their entirety.
[0099] In another embodiment, the human antibodies are raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. This mouse is referred to herein as a "KM Mouse®," and is described in detail in PCT Publication WO 02/43478. A modified form of this mouse, which further comprises a homozygous disruption of the endogenous FcRIIB receptor gene, is also described in PCT Publication WO 02/43478 and referred to herein as a "KM/FCGR2D Mouse®." In addition, mice with either the HCo7 or HCo12 heavy chain transgenes or both can be used.
[0100] Additional transgenic animal embodiments include the Xenomouse (Abgenix, Inc., U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963). Further embodiments include "TC mice" (Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727) and cows carrying human heavy and light chain transchromosomes (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894; PCT Publication WO 02/092812). The contents of these patents and publications are specifically incorporated herein by reference in their entirety.
[0101] Human monoclonal antibodies also can be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. See, e.g., U.S. Pat. Nos. 5,476,996 and 5,698,767, the contents of which are incorporated herein by reference in their entirety.
[0102] Antibodies of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
[0103] The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, e.g., in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma.
[0104] Alternatively, nonviral regulatory sequences can be used, such as the ubiquitin promoter or -globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al. (1988) Mol. Cell. Biol. 8:466-472). The expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
[0105] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In preferred embodiments, the variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
[0106] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
[0107] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because mammalian cells are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
[0108] Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, HEK293 cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
[0109] Thus, in another aspect, there is provided a cellular host that incorporates expressibly therein a polynucleotide that encodes the light chain, or the light chain variable region, of the misfolded human FasR antibody designated 3a-118, the polynucleotides having SEQ ID No. 101 (entire light chain) or SEQ ID No,102, respectively. In other embodiments, there is provided a cellular host that incorporates expressibly therein a polynucleotide that encodes the heavy chain, or the heavy chain variable region, of the misfolded human FasR antibody designated 3a-118, having SEQ ID No. 99 (entire heavy chain) or SEQ ID No. 100 (heavy chain variable region). In a further embodiment, there is provided a cellular host that incorporates expressibly therein both the heavy and light chain-encoding polynucleotides just recited, in either fully length form or in the form of variable region-encoding polynucleotides. In a still further embodiment, there comprises the step of culturing the transfected cellular host, thereby to produce the desired 3a-118 antibody.
[0110] Further, in embodiments, there is provided a cellular host that incorporates expressibly therein a polynucleotide that encodes the light chain, or the light chain variable region, of the misfolded human FasR antibody designated 3a-118, the polynucleotides having SEQ ID No. 101 (entire light chain) or SEQ ID No,102 In other embodiments, there is provided a cellular host that incorporates expressibly therein a polynucleotide that encodes the entire heavy chain SEQ ID No.99), or the heavy chain variable region (SEQ ID No.100), of the misfolded human FasR antibody designated 3a-118. In a further embodiment, there is provided a cellular host that incorporates expressibly therein both the heavy and light chain-encoding polynucleotides just recited, in either fully length form or in the form of variable region-encoding polynucleotides. In a still further embodiment, there comprises the step of culturing the transfected cellular host, thereby to produce the desired 3a-118 antibody.
[0111] Also, in embodiments, there is provided a cellular host that incorporates expressibly therein a polynucleotide that encodes the light chain, or the light chain variable region, of the misfolded human FasR antibody designated 3d-19, the polynucleotides having SEQ ID No. 115 (entire light chain) or SEQ ID No,116, respectively. In other embodiments, there is provided a cellular host that incorporates expressibly therein a polynucleotide that encodes the heavy chain, or the heavy chain variable region, of the misfolded human FasR antibody designated 3d-19, having SEQ ID No. 113 (entire heavy chain) or SEQ ID No. 114 (heavy chain variable region). In a further embodiment, there is provided a cellular host that incorporates expressibly therein both the heavy and light chain-encoding polynucleotides just recited, in either fully length form or in the form of variable region-encoding polynucleotides. In a still further embodiment, there comprises the step of culturing the transfected cellular host, thereby to produce the desired 3d-19 antibody.
[0112] Further, in embodiments, there is provided a cellular host that incorporates expressibly therein a polynucleotide that encodes the light chain, or the light chain variable region, of the misfolded human FasR antibody designated 3d-19, the polynucleotides having SEQ ID No. 115 (entire light chain) or SEQ ID No,116 In other embodiments, there is provided a cellular host that incorporates expressibly therein a polynucleotide that encodes the entire heavy chain SEQ ID No.113), or the heavy chain variable region (SEQ ID No.114), of the misfolded human FasR antibody designated 3d-19. In a further embodiment, there is provided a cellular host that incorporates expressibly therein both the heavy and light chain-encoding polynucleotides just recited, in either fully length form or in the form of variable region-encoding polynucleotides. In a still further embodiment, there comprises the step of culturing the transfected cellular host, thereby to produce the desired 3d-19 antibody.
[0113] For use in the methods of the present invention, the antibodies and their binding fragments can be conjugated with other agents that are useful for the intended purpose, e.g., either diagnostic use or medical treatment. Agents appropriate for treating disease include cytotoxic agents or toxins that include chemotherapeutics and radiotherapeutics. For diagnostic purposes, appropriate agents are detectable labels that include radioisotopes or fluorescent markers for whole body imaging, and radioisotopes, enzymes, fluorescent labels and the like for sample testing.
[0114] For diagnostics, the detectable labels can be any of the various types used currently in the field of in vitro diagnostics, including particulate labels including biotin/streptavidin, metal sols such as colloidal gold, isotopes such as 1125 or Tc99 presented for instance with a peptidic chelating agent of the N2S2, N3S or N4 type, chromophores including fluorescent markers such as FITC and PE, luminescent markers, phosphorescent markers and the like, as well as enzyme labels that convert a given substrate to a detectable marker, and polynucleotide tags that are revealed following amplification such as by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase and the like. For instance, the label can be the enzyme alkaline phosphatase, detected by measuring the presence or formation of chemiluminescence following conversion of 1,2 dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}deca- n}-4-yl)phenyl phosphate (CSPD), as well as CDP and CDP-Star® or other luminescent substrates well-known to those in the art, for example the chelates of suitable lanthanides such as Terbium(III) and Europium(III). The detection means is determined by the chosen label. Appearance of the label or its reaction products can be achieved using the naked eye, in the case where the label is particulate and accumulates at appropriate levels, or using instruments such as a spectrophotometer, a luminometer, a fluorimeter, and the like, all in accordance with standard practice.
[0115] For therapy, the cytotoxin can be conjugated with the antibody or binding fragment through non-covalent interaction, but more desirably, by covalent linkage either directly or, more preferably, through a suitable linker. In a preferred embodiment, the conjugate comprises a cytotoxin and an antibody. Immunoconjugates of the antibody and cytotoxin are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate, iminothiolane, bifunctional derivatives of imidoesters such as dimethyl adipimidate HCl, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates such as toluene 2,6-diisocyanate, and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Carbon-14-labeled 1-isothiocyanobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is a chelating agent suitable for conjugation of radionuclide to the antibody.
[0116] The cytotoxin component of the immunoconjugate can be a chemotherapeutic agent, a toxin such as an enzymatically active toxin of bacterial, fungal, plant or animal origin such as urease, or fragments thereof, or a small molecule toxin, or a radioactive isotope such as 212Bi, 131I, 131In, 111In, 90Y, and 186Re, or any other agent that acts to inhibit the growth or proliferation of a senescent cell.
[0117] Chemotherapeutic agents useful in the generation of such immunoconjugates include maytansinoids including DM-1 and DM-4, adriamycin, doxorubicin, epirubicin, 5-fluoroouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g. paclitaxel, and docetaxel, taxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins, 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycin D, VP-16, chlorambucil, melphalan, and other related nitrogen mustards. Also included are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone. Toxins and fragments thereof which can be used include diphtheria A chain, nonbonding active fragments of diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolaca Americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria, officinalis inhibitor, gelonin, saporin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothcenes. Small molecule toxins include, for example, calicheamicins, maytansinoids including DM-1 and DM-4, palytoxin and CC1065.
[0118] In an embodiment, the binding agent, optionally an antibody and/or binding fragment thereof, optionally conjugated to a toxin or a label or the nucleic acid is comprised in a composition. In an embodiment the composition comprises a diluent such as a saline solution for example phosphate buffered saline solution (0.05-1.0%).
[0119] The present invention also provides, for therapeutic use, a vaccine comprising any immunogenic form of an epitope that is unique to a misfolded form of a senescent cell surface protein, to treat subjects presenting with disease that is associated with senescent cells presenting that misfolded form of the protein. Such carriers will be nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the particular vaccine antigen with saline, buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose or dextrins, or chelating agents such as EDTA, glutathione and other stabilizers and excipients. Such compositions may be in suspension, emulsion or lyophilized form and will be pharmaceutically acceptable; i.e., suitably prepared and approved for use in the desired application. Preferred peptide vaccine compositions also comprise an adjuvant. DNA adjuvants are preferred for human use. The peptides may be formulated as fusions with other immunogenic peptides from the same or a different pathologic entity. Peptides may be synthesized as fusions of the epitopes identified herein with one or more T-helper epitope such as PADRE (SEQ ID NO: 118) or certain known tetanus peptides. Spacer peptides also may comprise part of these fusions. Materials having adjuvant activity are well known. Currently, however, Alum (Al(OH)3), and similar aluminium gels are the only adjuvants licensed for human use. Other materials are also known to have adjuvant activity, and these include: Freund's complete adjuvant, a water-in-mineral-oil emulsion which contains killed, dried mycobacteria in the oil phase; Freund's incomplete adjuvant, a weaker formulation without the mycobacteria; saponin, a membrane active glucoside extracted from the tree Quillia saponaria; nonionic block copolymer surfactants, non metabolised synthetic molecules which tend to bind proteins to cell surfaces; ISCOMS, lipid micelles incorporating Quil A (saponin) which mimic, in physical terms, infectious particles; and muramyl dipeptide, a leukocyte stimulatory molecule that is one of the active components of killed mycobacteria.
[0120] It will be appreciated that the vaccines noted above may comprise, instead of the epitope designated above, a variant thereof that incorporates 1, 2 or 3, amino acid additions, substitutions or deletions. Particularly the epitope may be a variant that has been truncated or extended to consist of 6, 7, or 8 amino acids, preferably 7 amino acids, and that incorporates up to 2, usually 1, amino acid substitution, for instance in which an amino acid is replaced by an oxidized form thereof, or an enantiomeric alternative thereof. It is apparent to those skilled in the art that substitution of certain amino acids in these epitopes will not affect immunoreactivity toward the epitopes. For example, substitution of leucine by isoleucine or valine and all combinations thereof is unlikely to alter the sensitivity of an antibody raised against this epitope. Thus all epitopes capable of generating antibodies reactive to the epitopes listed above for the purpose of selectively identifying misfolded specific protein are aspects of this invention.
[0121] It is occasionally desirable to derivatize amino acids present in the epitopes to obtain a more robust immune response or more selective reactivity toward the misfolded form. For example, a cysteine that on misfolding of its host protein may become oxidatively derivatized to cysteine sulfinic acid or cysteine sulfonic acid (cysteic acid). Thus antibodies against a free peptide containing, for example, a cysteic acid residue in place of cysteine are potentially more specific to the misfolded form of the protein. In general, candidate epitopes identified according to the methods described herein and containing derivatives of their constituent amino acids are an aspect of the present invention.
[0122] For epitopes containing proline, it may be desirable to prepare antigen peptides containing proline analogues that are fixed in the cis- or trans-configuration. Such analogues have been described previously (Scheraga et al, J Am Chem Soc 121 (49), 11558 (1999); Wang et al, J Org Chem 68 (6), 2343 (2002)). Unlike the other amino acids, for which there is a prohibitively large energy difference between the cis- and trans-amide bond stereoisomers, proline in unstructured peptides is able to interconvert between a cis- and trans-geometry on a relatively rapid time scale. When a proline is incorporated into the folded protein, steric interactions lock it into only one of the two possible conformers, but on unfolding it is free to racemerize. By raising antibodies against peptides incorporating a proline analogue with the opposite stereochemistry to that present in the native structure, the selectivity of the antibody for the unfolded state is much increased. Thus epitope peptides predicted by the method and incorporating cis- or trans-analogues of proline are an aspect of this invention.
[0123] In embodiments of the present invention, vaccines useful in the treatment of disease include the following:
a) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, and particularly 1, 2, 5 and 9, for the depletion of senescent cells presenting misfolded PrP protein; b) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 82, 83, 84, 85 and 86 for the depletion of senescent cells presenting misfolded CD38; c) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 47, 48, 49, 50 or 51 for the depletion of senescent cells presenting misfolded CD44; d) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 52, 53, 54, 55 or 56 for the depletion of senescent cells presenting misfolded Fas ligand; e) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 57, 58, 59, 60, or 61 for the depletion of senescent cells presenting misfolded NOTCH1; f) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 62, 63, 64, 65, and 66, and preferably SEQ ID Nos. 62 and 65, for the depletion of senescent cells presenting misfolded Fas receptor; and g) a vaccine that incorporates an immunogen comprising the epitope designated by SEQ ID No. 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 and 81 for the depletion of senescent cells presenting misfolded EGFR.
[0124] In therapeutic use, the antibodies and corresponding fragments and conjugates that bind selectively to the senescent cell marker, and vaccines that elicit such antibodies, can be used to treat subjects presenting with or at risk for a disease associated with cell senescence including particularly the so-called aging diseases and conditions, including degenerative disease. The terms "treat", "treatment," "treating", "therapeutic use," or "treatment regimen" encompass prophylactic, palliative, and therapeutic modalities of administration of the compositions of the present invention, and include any and all uses of the present products that remedy a disease state, condition, symptom, sign, or disorder caused or associated with, either directly or indirectly, a senescent cell presenting a misfolded form of a protein, including an inflammation-based pathology, infectious disease, allergic response, hyperimmune response, or other symptom to be treated, or which prevents, hinders, retards, or reverses the progression of symptoms, signs, conditions, or disorders associated therewith.
[0125] The term "subject" generally refers to mammals and other animals including humans and other primates, companion animals, zoo, and farm animals, including, but not limited to, cats, dogs, rodents, rats, mice, hamsters, rabbits, horses, cows, sheep, pigs, elk or other ungulates, goats, poultry, etc. A subject includes one who is to be tested, or has been tested for prediction, assessment or diagnosis of a disease or disorder associated with cell senescence. The subject may have been previously assessed or diagnosed using other methods, such as those in current clinical practice, or may be selected as part of a general population (a control subject). A subject may be a transgenic animal, e.g. a rodent, such as a mouse, that produces a target protein especially in misfolded form, or is lacking expression thereof (e.g. a `knock-out` mouse). For example, the subject may a transgenic mouse overexpressing a normal form of the target protein or may be a wild-type mouse or hamster that has been infected with a misfolded form of the target protein.
[0126] For treatment, the binding agent, such as the immunogen used for active immunization and the antibody used for passive immunization are used in "effective amounts". These are amounts useful, in a treatment regimen, to reduce the effect of the senescent cells that present the misfolded protein target. It will be apparent that the present invention is applicable to a wide variety of diseases, and that the particular amount and treatment regimen effective to reduce the effect of the endogenous protein will vary with each disease or condition, in accordance with established clinical practice.
[0127] In addition to such vaccines, the present invention provides for the therapeutic use of binding agents such as antibodies in the treatment of subjects presenting with the conditions noted above, including conditions/diseases related by the presence of the given misfolded protein. For treatment, antibody that binds selectively to the target epitope is administered as a pharmaceutical composition, comprising the antibody and a pharmaceutically acceptable carrier, in dosage form.
[0128] Also provided in another aspect, is a pharmaceutical composition comprising a binding agent that is selectively toxic to senescent cells in an amount effective to reduce the number of such cells for use in depleting senescent cells endogenous to a subject, wherein the binding agent binds selectively to a senescent cell surface protein having a misfolded conformation.
[0129] Yet a further aspect includes a pharmaceutical composition comprising a binding agent that selectively binds selectively to a senescent cell surface protein having a misfolded conformation.
[0130] For antibodies, fragments and conjugates, the dosage form is optionally a liquid dosage form. Antibody solutions can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose or an emulsifier such as polysorbate. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003-20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. Formulations optionally contain excipients including, but not limited to, a buffering agents, an anti-oxidant, a stabilizer, a carrier, a diluent, and an agent for pH adjustment. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum, albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG).
[0131] In treatment, the dose of antibody optionally ranges from about 0.0001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.15 mg/kg to about 3 mg/kg, 0.5 mg/kg to about 2 mg/kg and about 1 mg/kg to about 2 mg/kg of the subject's body weight. In other embodiments the dose ranges from about 100 mg/kg to about 5 g/kg, about 500 mg/kg to about 2 mg/kg and about 750 mg/kg to about 1.5 g/kg of the subject's body weight.
[0132] For example, depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of antibody or conjugate is a candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage is in the range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. Unit doses can be in the range, for instance of about 5 mg to 500 mg, such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg and 300 mg. The progress of therapy is monitored by conventional techniques and assays.
[0133] Therapeutic use of an antibody according to the present invention entails antibody administration, by injection or infusion, to subjects presenting with a disease in which cells or fluids present the epitope targeted by the antibody, i.e., in which the misfolded target protein is present. Subjects that would benefit from treatment can be identified by their clinical features, together with examination of tissue samples or bodily fluids to identify cells that present the epitope targeted by the antibody, as discussed infra.
[0134] In embodiments of the present invention, antibody compositions useful in the treatment of disease include compositions that incorporate the following:
a) an antibody that binds selectively to the epitope designated by SEQ ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, and particularly 1, 2, 5 and 9, for the depletion of senescent cells presenting misfolded PrP protein; b) an antibody that binds selectively to the epitope designated by SEQ ID No. 82, 83, 84, 85 and 86 for the depletion of senescent cells presenting misfolded CD38; c) an antibody that binds selectively to the epitope designated by SEQ ID No. 47, 48, 49, 50 or 51 for the depletion of senescent cells presenting misfolded CD44; d) an antibody that binds selectively to the epitope designated by SEQ ID No. 52, 53, 54, 55 or 56 for the depletion of senescent cells presenting misfolded Fas ligand; e) an antibody that binds selectively to the epitope designated by SEQ ID No. 57, 58, 59, 60, or 61 for the depletion of senescent cells presenting misfolded NOTCH1; f) an antibody that binds selectively to the epitope designated by SEQ ID No. 62, 63, 64, 65, and 66, and preferably SEQ ID Nos. 62 and 65, for the depletion of senescent cells presenting misfolded FasR; and g) an antibody that binds selectively to the epitope designated by SEQ ID No. 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 and 81 for the depletion of senescent cells presenting misfolded EGFR.
[0135] For treatment with a vaccine, subjects are immunized on a schedule that can vary from once a day, to once a week, to once a month, to once a year, to once a decade. A typical regimen includes an immunization followed by booster injections at 6 weekly intervals. Another regimen consists of immunization followed by booster injections 1, 2 and 12 months later. Alternatively, booster injections will vary depending on the immune response and the physiological condition of the subject. For immunization, the epitope-containing immunogen can be administered in a dose that ranges from about 0.0001 microgram to 10 grams, about 0.01 microgram to about 1 gram, about 1 microgram to about 1 mg, and about 100 to 250 micrograms per treatment. In one embodiment the timing of administering treatment is at one or more of the following: 0 months, 2 months, 6 months, 9 months, and/or 12 months. In one regimen, the dosing is at 2, 6, 9, and 12 months following the first immunization. In another regimen, the dosing is at 2 and 4 weeks following the first immunization, and then monthly afterwards. In an alternative regimen, the dosing varies depending on the physiological condition of the subject and/or the response to the subject to prior immunizations. The route of administration optionally includes, but is not limited to, intramuscular and intraperitoneal injections. In one embodiment the composition is injected into the deltoid muscle.
[0136] The vaccine composition itself can further comprise adjuvants. Adjuvants for parenteral immunization include aluminum compounds (such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate). The antigen can be precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants such as RIBI (ImmunoChem, Hamilton, Mont.) can also be used in parenteral administration.
[0137] The active ingredients to be used for in vivo administration will be sterile. This is readily accomplished by filtration through sterile filtration membranes.
[0138] Sustained-release preparations may be prepared. Suitable examples of sustained-release include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shapes articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate, and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
[0139] Diagnostically useful compositions comprising the antibody will incorporate a carrier suitable for diagnostic purposes, such as a solution of saline or buffered saline including phosphate buffered saline, together with any desired stabilizers or preservatives. Of course, the composition can be provided in a lyophilized form to prolong storage stability.
[0140] In diagnostic use, the binding agent is exploited to detect the presence of senescent cells. The senescent cells to be detected may be in a sample obtained from any source, including a living subject. A subject includes one who is to be tested, or has been tested for prediction, assessment or diagnosis of a disease or disorder associated with a given misfolded protein target. The subject may have been previously assessed or diagnosed using other methods, such as those in current clinical practice, or may be selected as part of a general population (a control subject). A subject may be a transgenic animal, e.g. a rodent, such as a mouse, that produces a senescent cell presenting a target protein in misfolded form. For example, the subject may be a transgenic mouse overexpressing a normal form of the target protein or may be a wild-type mouse or other rodent that has been infected with a misfolded form of the target protein.
[0141] To assist with the identification of subjects who are candidates for treatment with the antibody or vaccine compositions of the invention, the present invention further provides for the detection of an epitope by in vitro or in vivo diagnostic methods.
[0142] To detect the presence of a misfolded protein in any given sample, the present invention provides a detection method in which a sample suspected to contain the misfolded protein is treated with an antibody or binding fragment that binds selectively to an epitope presented uniquely by the misfolded protein relative to the natively folded form of that protein; and determining whether an antigen:antibody complex has formed, the formation thereof being indicative of the presence in the sample of a misfolded form of said protein. In one embodiment, the epitope is one that has been identified by applying an epitope prediction method. In another embodiment, the epitope is one that is, or is comprised within, a peptide having any one of SEQ ID Nos.1-36 and 41-86.
[0143] In a related embodiment, the labeled antibodies of the invention, or labeled form of a binding fragment thereof, can be used in vivo to image the presence of the misfolded protein to which the antibody binds. To this end, the present invention provides an antibody or fragment in a form coupled to an agent useful for in vivo imaging, such as isotopes of technetium, gadolinium, and the like.
[0144] In situ detection of the binding to cancer cells bearing misfolded PrP can also be performed using the present antibody or fragment, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological specimen is removed from the patient, and a labeled form of the present antibody is applied to it, preferably by overlaying the antibody on a biological sample, in keeping with standard immunohistochemistry techniques. This procedure also allows for distribution of the PrP antigen to be examined within biopsied tumour tissue, to reveal only those sites at which PrP is presented in misfolded form. It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in situ detection.
[0145] More particularly, antibodies or binding fragments of the present invention may be used to monitor the presence or absence of antibody reactivity in a biological sample, e.g., a tissue biopsy from brain, skin, liver, heart, kidney, pancreas, bowel, spleen, muscle, fat, skin, ovary and the like, from a cell, or from fluid such as cerebrospinal fluid, blood including plasma, urine, seminal fluid, and the like, using standard detection assays. Immunological assays may involve direct detection, and are particularly suited for screening large amounts of samples for the presence of senescent cells that present misfolded surface protein. For example, antibodies may be used in any standard immunoassay format (e.g., ELISA, Western blot, immunoprecipitation, flow cytometry or RIA assay) to measure complex formation. Any appropriate label which may be directly or indirectly visualized may be utilized in these detection assays including, without limitation, any radioactive, fluorescent, chromogenic (e.g., alkaline phosphatase or horseradish peroxidase), or chemiluminescent label, or hapten (for example, digoxigenin or biotin) which may be visualized using a labeled, hapten-specific antibody or other binding partner (e.g., avidin). Exemplary immunoassays are described, e.g., in Ausubel et al., supra, Harlow and Lane, Antibodies: A Laboratory Approach, Cold Spring Harbor Laboratory, New York (1988), and Moynagh and Schimmel, Nature 400:105, 1999. For example, using the antibodies described herein, misfolded PrP is readily detected at the cell surface using standard flow cytometry methods. Samples found to contain labeled complex compared to appropriate control samples are taken as indicating the presence of misfolded PrP, and are thus indicative of a disease state amenable to treatment with the present antibodies.
[0146] Senescent cell screening results that are obtained with misfolded protein binding agents can be confirmed using any other test appropriate for senescent cell detection. It is known, for instance that misfolded PrP is detectable not only on senescent cells, but also on certain cancer cells particularly including ovarian cancer cells. In certain instances, therefore, the present senescent cell detection method should be accompanied by a test that is confirmatory for senescent cells. In embodiments, the confirmatory test can be the established assay for B-galactosidase activity. It has been shown that senescent cells will use B-galactosidase as a substrate when cultured at mildly acidic pH, such as pH 6.0. As described by Dimri et al in U.S. Pat. No. 5,795,728, incorporated herein by reference, senescent cells can be identified by culturing cells in the presence of a B-galactosidase substrate, such as X-gal, and at pH 6.0. The cultured cells or tissue are then fixed in a solution such as 2% formaldehyde, 0.2% glutaraldehyde and PBS. The appearance of reaction products, revealed by staining, indicates that the cultured cell is senescent. Alternatively, any cell or tissue can be examined for senescence by assaying for the presence of INK4a/ARF expression, as described by Sharpless et al in U.S. Pat. No. 8,158,347, also incorporated herein by reference. An elevation in this expression product indicates the tested cell is senescent. Of course, other methods are useful to identify senescent cells, including measurement of the incorporation of labeled DNA precursors such as 3H-thymidine and BrdU or measurement of cell markers that are expressed only in proliferating cells, such as PCNA or MTT. Senescent cells will test negative for these markers.
[0147] Such assays can also be performed prior to administering a binding agent or immunogen described herein and/or to identify subjects to be treated. In an embodiment, the method comprises detecting senescent cells in a sample containing cells to be screened according to a method described herein, for example using a binding agent, B-galactosidease assay etc or combinations thereof; and administering to the subject a binding agent that is selectively toxic to senescent cells in an amount effective to reduce the number of such cells, wherein the biding agent binds selectively to a senescent cell surface protein having a misfolded conformation, relative to said protein in a natively folded conformation. In an embodiment, the method comprises detecting senescent cells in a sample containing cells to be screened according to a method method described herein, for example using a binding agent, B-galactosidase assay etc or combinations thereof; administering to the subject a binding agent that binds selectively to a senescent cell surface protein having a misfolded conformation, for example to treat an age-related disease.
[0148] To detect the presence of a senescent cell in a given sample, the present invention provides a detection method in which a sample suspected to contain the senescent cell is treated with a binding agent, e.g., an antibody or binding fragment, that binds selectively to an epitope presented uniquely by the misfolded protein relative to the natively folded form of that protein; and determining whether an antigen:antibody complex has formed, the formation thereof being indicative of the presence in the sample of a senescent cell. In one embodiment, the epitope is one that is, or is comprised within, a peptide herein described.
[0149] When applied in vitro, the detection method entails analysis of a cell-containing sample of body fluid or tissue or organ from a subject, usually a subject suspected of having endogenous misfolded target protein. For example, the biological sample may a body fluid such as cerebrospinal fluid, blood, plasma, lymph fluid, serum, urine or saliva. A tissue or organ sample, such as that obtained from a solid or semi-solid tissue or organ, may be digested, extracted or otherwise rendered to a liquid form--examples of such tissues or organs include cultured cells, blood cells, brain, neurological tissue, skin, liver, heart, kidney, pancreas, islets of Langerhans, bone marrow, blood, blood vessels, heart valve, lung, intestine, bowel, spleen, bladder, penis, face, hand, bone, muscle, fat, cornea or the like, including cancerous forms thereof. A biological sample or samples may be taken from a subject at any appropriate time, including before the subject is diagnosed with, or suspected of having a protein misfolding associated disease or disorder, during a therapeutic regimen for the treatment or amelioration of symptoms of that disease or disorder, after death of the subject (regardless of the cause, or suspected cause). Alternately, a biological sample may include donated body fluid or tissue, such as blood, plasma or platelets when in care of a centralized blood supply organization or institution.
[0150] The presence in the sample of a senescent cell presenting a misfolded target protein is confirmed if the antibody forms a detectable antigen:antibody complex. The formation of such complex can be determined using a wide variety of protocols that include ELISA, RIA, flow cytometry, Western blots, immunohistochemistry and the like. To reveal the complex and hence the presence of the epitope in the sample, the antibody desirably is provided as a labeled antibody by conjugation or coupling to an agent that is detectable either visually or with the aid of instrumentation. The agent, or label, is capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. Alternatively, the epitope can be revealed using a labeled secondary reagent that binds to the epitope antibody, such as a labeled antibody that binds the epitope antibody, to reveal presence of the epitope indirectly. The presence of an antibody:antigen complex may be detected by indirect means that do not require the two agents to be in solution. For instance, the complex is detectable indirectly using flow cytometry, where the antibody binds to, and forms an antibody:antigen complex with, the epitope presented on the surface of an intact cell. The application of the antibodies for detection of cell-surface forms of the epitope is a very useful embodiment of the invention particularly for detection of senescent cells presenting such epitopes. Detection of such cells can be achieved using the well-established technique of flow cytometry. It will also be appreciated that the antigen:antibody complex can also be identified by non-antibody based methods, that include those which sort proteins based on size, charge and mobility, such as electrophoresis, chromatography, mass spectroscopy and the like.
[0151] In a related embodiment, the labeled antibodies of the invention, or labeled form of a binding fragment thereof, can be used in vivo to image the presence of senescent cells that present the misfolded protein to which the antibody binds. To this end, the present invention provides an antibody or fragment in a form coupled to an agent useful for in vivo imaging, such as isotopes of technetium, gadolinium, and the like. In embodiments, the senescent cell detection method is conducted using antibodies that bind selectively to misfolded human PrP. In specific embodiments, the antibodies are selected from 1A1, c-120 and YYR MAbs, and misfolded PrP binding fragments and conjugates thereof. In other specific embodiments, the antibodies are selected from 3d-19 and 3a-118, and misfolded FasR-binding antibody fragments and conjugates thereof.
[0152] In therapeutic use, the binding agents that bind selectively to a misfolded protein marker on the surface of senescent cells can be used to treat patients or subjects presenting with or at risk for a disease associated with senescent cells. For therapeutic use, passive immunotherapy can be adopted by administering binding agents that are antibodies or binding fragments thereof. In the alternative, active immunotherapy can be adopted using vaccines that elicit the production of such antibodies.
[0153] For treatment, the active ingredient, such as the immunogen used for active immunization and the antibody used for passive immunization are used in "effective amounts". These are amounts useful, in a treatment regimen, to reduce the effect of the endogenous senescent cells by eliminating or reducing the number of senescent cells endogenous to the recipient. It will be apparent that the present invention is applicable to a wide variety of diseases, and that the particular amount and treatment regimen effective to reduce the effect of the endogenous protein will vary with each disease, in accordance with established clinical practice for each disease.
[0154] An anti-senescence therapeutic according to the invention may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
[0155] Any appropriate route of administration can be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
[0156] For the treatment of subjects presenting with senescent cells positive for misfolded target protein, the appropriate dosage of agent, e.g., an antibody, fragment or conjugate, will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventative or therapeutic purposes, previous therapy, the patients clinical history and response to the agent, and the discretion of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments, in accordance with dosing regimens discussed above.
Indications
[0157] It is anticipated that depletion of endogenous senescent cells will have a therapeutic effect and benefit on a variety of aging diseases and conditions, including particularly the degenerative disorders that accompany aging. More particularly, senescent cell depletion is expected to provide improvements in 1) reducing the rate at which adipose tissue is lost, 2) reducing the rate at which muscle fibre diameter is reduced, and 3) reducing the rate at which skin tone deteriorates over time. These effects are likely to be seen more dramatically in aged recipients, i.e. those at an age greater than 50 years, especially those aged greater than 60 years or more, such as 65 years, 70 years and 75 years and greater. Also, candidate recipients include those whose lifestyle imposes age-accelerating effects, including tobacco smokers and users, alcohol and narcotic drug abusers, skin tanning enthusiasts, and the like. These are therefore preferred recipients of the present treatment method.
[0158] Particular conditions and diseases that can be treated by the present method include sarcopenia. Sarcopenia is characterized first by a muscle atrophy (a decrease in the size of the muscle), along with a reduction in muscle tissue "quality," caused by such factors as replacement of muscle fibres with fat, an increase in fibrosis, changes in muscle metabolism, oxidative stress, and degeneration of the neuromuscular junction. Combined, these changes lead to progressive loss of muscle function and frailty.
[0159] Other conditions that can be treated by the present method include cataracts, and so-called "signs of aging" such as wrinkling and discolouration of the skin, and overall dermal tone. Treatment by the present method is expected to reduce the rate at which fat and muscle that support skin tone are reduced, so that skin wrinkling also is reduced, delayed or eliminated. As well treatment is expected to have a benefit on the rate at which cataracts form in the eye.
[0160] It has been demonstrated that senescent cells create a local environment that supports tumorigenesis in neighbouring cells. Treatment by the present method can accordingly be useful to treat cancer in various tissues, including cancers of the lung, prostate, skin, breast, and the like. Such treatment is expected to result in a decrease in the rate of tumour formation directly, and thus indirectly also on the number, size and distribution of responsive cancer cells and tumours.
[0161] In specific embodiments of the present invention, antibody compositions useful in the treatment of disease include compositions that incorporate the following:
a) an antibody that binds selectively to an epitope presented by peptides designated by SEQ ID No. 1-37 and 47-86 for the treatment of subjects afflicted with sarcopenia; and b) an antibody that binds selectively to an epitope presented by peptides designated by SEQ ID No. 1-37 and 47-86 for the treatment of aged subjects presenting with signs of skin aging such as wrinkling, discolouration and the like; and c) an antibody that binds selectively to an epitope presented by peptides designated by SEQ ID No. 1-37 and 47-86 for the treatment of aged subjects presenting with tumours.
[0162] The invention also includes articles of manufacture as well as kits that comprise components useful to perform the diagnostic and therapeutic methods of the present invention. The articles of manufacture comprise packaging material and a composition comprising an antibody or antisera that binds selectively to senescent cells that present an epitope unique to a misfolded form of protein, relative to the natively folded form of that protein. The composition includes a physiologically or pharmaceutically acceptable excipient, and the packaging material may include a label which indicates the active ingredients of the composition (e.g. the antisera or antibody). The label may further include an intended use of the composition, for example as a diagnostic reagent to be used with kits as set out herein.
[0163] Also provided is an article of manufacture, comprising packaging material and a composition comprising a peptide, or one or more peptides, as provided herein. The composition may include a physiologically or pharmaceutically acceptable excipient, and the packaging material may include a label which indicates the active ingredients of the composition (e.g. the peptide). The label may further include an intended use of the composition, for example as a therapeutic or prophylactic reagent, or as a composition to induce an immune response in a subject for the purpose of producing antisera or antibodies specific to senescent cell target protein, to be used with kits as set out herein.
[0164] In a further embodiment, there is provided a kit comprising a composition comprising one or more peptides as provided herein, along with instructions for use of the compound or composition for the production or screening of antibodies for identification of senescent cells. The kit may be useful for production and/or identification of senescent cell-specific antibodies or antisera, and the instructions may include, for example, dose concentrations, dose intervals, preferred administration methods, methods for immunological screening or testing, or the like.
[0165] In another embodiment, a kit for the preparation of a medicament, comprising a composition comprising one or more peptides as provided herein, along with instructions for its use is provided. The instructions may comprise a series of steps for the preparation of the medicament, the medicament being useful for inducing a therapeutic or prophylactic immune response in a subject to whom it is administered. The kit may further comprise instructions for use of the medicament in treatment for treatment, prevention or amelioration of one or more symptoms of a disease or disorder associated with protein misfolding on senescent cells, or in which protein misfolding is implicated, and include, for example, dose concentrations, dose intervals, preferred administration methods or the like.
[0166] In another embodiment, a kit for diagnosing a disease or disorder associated with protein misfolding is provided. The kit comprises one or more misfolded protein-selective antibodies or antisera as described herein, along with instructions for its use. The antibody may further be coupled to a detection reagent. Examples of detection reagents include secondary antibodies, such as an anti-mouse antibody, an anti-rabbit antibody or the like. Such secondary antibodies may be coupled with an enzyme that, when provided with a suitable substrate, provides a detectable colorimetric or chemiluminescent reaction. The kit may further comprise reagents for performing the detection reaction, including enzymes such as proteinase K, blocking buffers, homogenization buffers, extraction buffers, dilution buffers or the like. The kit may further comprise reagents useful to perform the confirmatory B-galactosidase test for senescence, as discussed above.
[0167] In another embodiment, a kit for detecting the presence of senescent cells in a biological sample is provided. The kit comprises one or more antibodies or antisera that specifically bind the misfolded protein presented by the senescent cells, along with instructions for its use. The antibody may further be coupled to a detection reagent. Examples of detection reagents include secondary antibodies, such as an anti-mouse antibody, an anti-rabbit antibody or the like. Such secondary antibodies may be coupled with an enzyme that, when provided with a suitable substrate, provides a detectable colorimetric or chemiluminescent reaction. The kit may further comprise reagents for performing the detection reaction, including enzymes such as proteinase K, blocking buffers, homogenization buffers, extraction buffers, dilution buffers or the like. The kit may further comprise reagents useful to perform the confirmatory B-galactosidase test for senescence, as discussed above.
Example 1
Antibodies to YYR Epitope of Misfolded Human PrP (See U.S. Pat. No. 7,041,807)
[0168] Epitopes uniquely exposed on misfolded, but not natively structured, PrPC reside in the peptides RYYRENMH and QVYYRPV (SEQ ID Nos 5 and 7 respectively). Resident within each of these peptides is the 3-amino acid epitope designated YYR noted above. Antibodies against the YYR epitope of PrP are described in the literature by Cashman (see U.S. Pat. No. 7,041,807 incorporated herein by reference). Some of that work is reproduced below. As noted in the results, the antibodies to YYR bind selectively to the disease-misfolded form of PrPC, and do not bind to the natively folded form.
[0169] In order to develop an antibody to the YYR epitope presented by misfolded PrPC and but not by natively folded PrPC, a peptide with the amino acid sequence Acetyl-Cys-Tyr-Tyr-Arg-NH2 (YYR) was synthesized, conjugated to KLH, and injected intramuscularly into rabbits using well known techniques. At the amino-terminus of the peptide, a cysteine residue was added to allow conjugation of the peptide with the protein carrier. The amino group of the peptide was blocked by acetylation, and the carboxylic group of the peptide was blocked by amidation.
[0170] Peptides were synthesized using solid phase peptide synthesis methods either manually or automated (MPS396 peptides synthesizer, Advanced ChemTech). Coupling of amino acid residues was accomplished using Fmoc peptide synthesis chemistry (Fields et al., 1990, IJPPR 35, 161). Syntheses were performed on Wang or on amide Rink resins, with full side chain protection of amino acids. Since the alpha-NH2 groups of the amino acids were protected with the Fmoc group, the following protective groups were chosen for the side groups of the trifunctional amino acids:
Cysteine: 5-triphenylmethyl (Trt) Arginine: 2,2,4,6,7-pentamethyldihydrobenzofuran-5 sulfonyl (Pbf) Tyrosine: tert.-butyl ether (tBu)\
[0171] BOP, PyBOP, or TBTU were used as activation agents, depending on the chemistry and difficulty of the coupling reaction. All chemicals were purchased from Advanced Chem Tech, Bachem, and Calbiochem/NovaBiochem. Formation of each peptide bond between residues of the sequence was ensured by using a 3 to 6 fold excess of coupling reagents and by so-called double coupling; meaning that the coupling reaction was repeated for each amino acid added to the growing peptide chain.
[0172] After synthesis, the peptides were cleaved from the resin using the Reagent K as a cleavage mixture: water (2.5%), TIS (2.5%), EDT (2.5%), TFA (92.5%). The peptides were then precipitated with cold diethyl ether. The precipitates were centrifuged, washed three times with diethyl ether, dissolved in 20%-50% AcCN/water mixture, and lyophilized. Analysis of crude products was performed using analytical RP-HPLC and electrospray MS.
[0173] The crude peptide was purified by Rp-HPLC (reverse phase high performance liquid chromatography) on a Vydac C18 column, 2.5×25 cm, using a linear gradient of 10-50% acetonitrile in water, with 0.06% TFA (1%/min gradient, 10 ml/min flow rate), with monitoring by UV at 215 nm and 254 nm. Analytical HPLC was used to estimate the purity of the fractions. The final product was obtained as a lyophilized peptide with at least 95% purity estimated by analytical HPLC (Vydac C18, 0.46×25 cm, linear gradient 10-60% acetonitrile in water, 0.1% TFA, 1%/min, 1 mL/min flow rate, detection by UV absorption at 215 nm and 254 nm). The pure peptide was identified by molecular mass analysis using a SCIEX API III mass spectrometer according to standard procedures.
[0174] The retention time of the peptide on RP-HPLC was 21.215 minutes. The theoretical molecular weight of the peptide was calculated to be 644.74; the actual molecular weight, through molecular mass analysis, was found to be 646.5 (MW+H*).
[0175] Peptides were coupled to a carrier, in this case Keyhole limpet hemocyanin (KLH). Other carriers useful for such coupling include, without limitation, albumin, or ovalbumin, 8map, or lysozyme. Coupling was effected via a thioether linkage to the mercapto group of the cysteine. This type of linkage has the advantage that the peptide is coupled in a defined way to a carrier protein.
[0176] Coupling to KLH was performed as follows. 10 mg of the peptide was dissolved in 2 ml of phosphate buffered solution (PBS 1×). 1 ml of KLH (pierce products #77100) was added to the peptide solution and stirred (1 mole of peptide/50 amino acids). The KLH concentration was 10 mg/ml. 20 ul of glutaraldehyde (25% aqueous solution) was added to the peptide/carrier solution with constant stirring, incubated for 1 hour, after which a glycine stop solution was added. The peptide/carrier conjugate was separated from the peptide by dialysis against PBS
[0177] Polyclonal antibodies were prepared according to standard methods, and an immune response was enhanced with repeated booster injections, at intervals of 3 to 8 weeks. The success of the immunization was verified by determining the concentration of antibodies in a western blot or ELISA or both. More specifically, to generate polyclonal antibodies to misfolded PrPC (or PrPSc), the tripeptide YYR conjugated to KLH was injected into rabbits in accordance with a 164 day immunization regimen, after which the animals that had produced specific antibodies were bled.
[0178] In order to sample the serum prior to immunization, 10 ml of blood per rabbit was taken as a preimmune control. Primary immunizations were carried out with Freund's complete adjuvant and subsequent boosts with incomplete Freund's adjuvant (IFA0 (1 ml per rabbit, 0.5 ml per thigh muscle). Each injection consisted of approximately 200 ug of the purified peptide. At days 21, 42 and 70, a booster injection was given with IFA. At days 31, 42 and 80, 10 ml of blood was collected from the central ear artery for titer determination (6 ml/kg/rabbit). At day 80, the titer of the sera was checked, and 3 more injections were given (IFA) at 4 week intervals, followed by blood sampling 10 days later. 10 days after the last boost, anesthetized rabbits were exsanguinated via cardiac puncture, and antisera were collected.
[0179] Goat polyclonal antibodies were generated according to standard methods. Three goats were immunized as follows. On day 1, all the goats received a primary immunization of 1 mg of YYR-KLH conjugates in complete Freund's adjuvant. Boosts were done by injection of 1 mg YYR-KLH in incomplete Freund's adjuvant for two of the three goats, whereas the third goat received 1 mg YYR-8map conjugates in incomplete Freund's adjuvant. Serum samples from each of the three bleeds were tested for reactivity by ELISA against YYR-BSA conjugates. From the third set of bleeds, total IgG was purified by ammonium sulfate precipitation and YYR-reactive IgG was purified using a YYR affinity column. IgG fractions were tested for reactivity to PrPSc as described herein. The exact immunization schedule was as follows: Day 1, primary immunization; D 21, first boost immunization; Day 30, first bleed; Day 46, second boost immunization; Day 53, second boost immunization; Day 60, second bleed; Day 76, third boost immunization; Day 83, third boost immunization; and Day 90, third bleed.
[0180] Alternatively, monoclonal antibodies may be prepared using the synthetic peptides described herein and standard hybridoma technology (see, e.g., Kohler et al., Nature 256, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976 Hammerling et al., In Monoclonal Antibodies and T Cell Hydridomas, Elsevier, NY, 1981; Ausubel et al., 1999, Current Protocols in Molecular Biology, Wiley Interscience, New York,) Once produced, monoclonal antibodies are also tested for specific PrP recognition by immunoprecipitation and western blot analysis.
[0181] The generation of monoclonal antibodies was carried out as follows. Mice were immunized with baculovirus supernatant containing mouse PrP-AP fusion protein in complete Freund's adjuvant, then boosted 2 weeks later with the same antigen in incomplete Freund's adjuvant. Two weeks after that immunization the mice were boosted with a mixture of PrP-AP supernatant plus 100 ug of KLH-CYYRRYYRYY (SEQ ID NO: 90 and 10 ug of KLH-CKYEDRYYRE (SEQ ID NO: 91) conjugates. Splenocytes from these mice were fused to the FO murine B cell line (ATCC CRL-1646) to generate specific hybridoma clones. Hybridoma supernatants were screened by ELISA. There were no reactive supernatants to PrP-AP or to the CKYEDRYYRE (SEQ ID NO: 91) sequence, although there were clones reactive to YYR-8map conjugates.
[0182] Total rabbit IgG was purified from serum using the Pharmacia protein A HiTrap column according to the manufacturer's recommendations. Briefly, a HiTrap column was equilibrated with 3 column volumes of start buffer (0.2M sodium phosphate buffer, pH7.0). Serum was applied, using a syringe through a luer adaptor, onto the column. The column was subsequently washed with 5 ml of start buffer. Bound protein was eluted with 0.1M glycine, pH 3.0, and collected in eppendorf tubes containing 1M Tris pH 8.0 (50 ul/500 ul sample). Fractions were analyzed on SDS-PAGE.
[0183] Goat polyclonal antibodies were purified from serum samples as described above.
[0184] Mouse monoclonal antibodies were produced as ascites, and purified using a protein A column kit (Pierce) according to the manufacturer's instructions. Briefly, a sample of ascites was diluted with binding buffer at a 1:1 final ratio. The sample was then added to the top of the column, which had been previously equilibrated with binding buffer, and allowed to flow through the matrix. The pass-through material was collected and the column washed with 5 volumes of binding buffer. Mild elution buffer was added to the column to release the bound IgG antibody from the matrix. Other antibody isotypes were collected by switching to the IgG elution buffer. All the antibodies were collected in 1 ml fractions, which were analyzed by BCA to determine total protein content and SDS-PAGE electrophoresis to establish the degree of antibody purity. The fraction containing the most yield of IgG was desalted by passing it through a D-salt column (Pierce). The antibody fraction was allocated and stored at -80 C. in PBS
[0185] Antibodies produced using the aforementioned procedures were subsequently tested for high-affinity binding as follows.
[0186] Ten ul of brain extract was added to 950 ul of Immunoprecipitation buffer (PBS 3% NP-40, 3% Tween-20) and incubated at 37° C. for 30 or 60 minutes. For experiments evaluating the reactivity of PrP 27-30 with the bead conjugates, the incubation was preceded by addition of 50 ul of 1 mg/ml proteinase K. Samples not treated with proteinase K were still incubated at 37 D. for the appropriate time period. After the incubation, 60 ul of a 100 mM PMSF solution were added to both sets of tubes. On hundred ul of resuspended bead conjugates were then added to the mixture, and incubated with rotation at room temperature for 2 hours. The beads were washed 3 times with washing buffer (PBS 2% NP-40 2% Tween-20) and resuspended by vortex after each wash. After the last wash, the beads were resuspended in 20 ul of 2× loading buffer (100 mM Tris pH 6.8, 4% SDS, 0.015% bromphenol blue, 20% glycerol) and heated at 95° C. for 3 minutes.
[0187] The PrPSc content of brain homogenates was determined by western blotting according to standard methods. Protein samples were mixed with 2× sample buffer at a ratio of 1:1 and boiled for 5 minutes at 100° C. SDS-PAGE analysis was performed according standard methods. Samples were applied to a pre-cast 15% acrylamide gels (Biorad) along with pre-stained molecular weight markers (Biorad). The gels were run at 100 V until the bromophenol blue dye front reached the bottom of the gel. The separated protein was then transferred onto PVDF membranes at 100 V or 1 hr. The membranes were washed as described above before incubation with a goat anti-mouse IgG alkaline phosphatase conjugated secondary antibody (1:5000 in TBST) for 1 hour at room temperature. After washing, signals were developed with the chemiluminescent substrate CDP-star, and exposed to x-ray films.
[0188] Spleen cell suspensions were prepared from Balb/c mice by passing the tissues through a wire mesh. The cells were washed once with cold Dulbecco's PBS without Ca2+ or Mg2+ and viable cells were isolated by underlayering of the cell suspension with Lympholyte (Cedarlane) and centrifugation at 1300 g for 20 minutes. The cells were washed once with cold Dulbecco's PBS without CA2+ or Mg2+2.5% fetal bovine serum, and 0.5×106 cells were aliquoted per well in a round bottom 96 well plate. The cells were centrifuged and resuspended in 50 ul of antibody-FITC conjugates at 1/10 final concentration in Dulbecco's PBS without CA2+ or Mg2+2.5% fetal bovine serum, for 15 minutes on ice. The cells were then washed twice with cold Dulbecco's PBS without Ca2+ or Mg2+2.5% fetal bovine serum and resuspended in the same medium containing 1 ug/ml of propidium iodide. The cells were analyzed on a Coulter Epics flow cytometer and were gated by size and granularity (forward and side scatter) and viability (exclusion of propidium iodide fluorescence).
[0189] Fluoresceinated mAbs were made by using the Fluorotag kit (Sigma) following the manufacturer's instructions. Briefly, 0.5 mg of each antibody was raised to pH 9 with concentrated bicarbonate buffer, and FITC stock solution was added to produce an FITC: antibody ratio of 20:1. The vials were then incubated for 2 hours at room temperature. Labeled antibody was separated from free FITC by passing the mixture over a Sephadex G-25M column. Conjugated antibodies were tested for successful fluoresceination by measuring their FITC emissions at 35 nm using an LJL Biosystems Analyst, and the antibodies were tested for retention of their binding activity with an ELISA against YYR-8map conjugates.
[0190] To determine whether antibody pAbC2 was useful in specifically recognizing PrPSc from bovine brain extracts, compared to PrPC using recombinant PrP (rbPrP), an ELISA approach was used. Either pools of PrPSc containing brain extracts or rbPrP was used to test the specificity of pAbC2 for PrPSc. The wells of an Immunolon ELISA plate (Dynex) were coated overnight at 4° C. with the PC2 containing culture supernatant in a TBS buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl2. For BSE-brain extract experiments, control wells were coated with a supernatant containing Mek-4; for rbPrP experiments, milk was used as a control to determine the non-specific binding of the antibody to the well. The coating of the ELISA plates with soluble PC2 was confirmed with an anti-PC2 monoclonal antibody. The wells were washed four times using a SLT `Columbus` microplate washer (Tecan) with TBS containing 0.05% Tween 20, and blocked by filling the wells with 0.2% I-Block (Tropix) in TBST and incubating the plate at 37° C. for 1 hour. The plates were washed and the bovine brain homogenate (diluted to 1% w/v in TBS) or rbPrP was added to designated wells and incubated at RT for 1 h. Wells were washed four times with TBST. pAbC2 was added to appropriate wells and incubated at RT or 1 hour, followed by a further 45 minute incubation with 100 ul of an anti-rabbit or mouse IgG/horseradish peroxidase conjugate (1:5000) in TBST containing 1% non-fat milk. Wells were washed four times with TBST. Signals were developed with TMB/H2O2 as a substrate for peroxidase. Reactions were stopped after 15 minutes by the addition of 100 ul of 2M phosphoric acid. Signals were monitored at 450 nm with reference at 620 nm using a SLT microplate reader. Specific positive signals were determined by comparing PrP binding to PC2 with PrP binding to the negative control, Mek-4 or milk. Preimmune controls showed no binding.
[0191] It will thus be appreciated that various antibodies that recognize and bind selectively to the YYR epitope unique to misfolded PrPC can be obtained by raising antibodies against those epitopes in accordance with standard practices well established for these purposes.
Example 2
Antibodies to YML Epitope of Misfolded Human PrP (See WO 2010/099612)
[0192] As shown in Table 1, a region of PrPC beta strand 1 contains the epitope designated YML as shown by peptides LGGYML and GGYMLGS of SEQ ID Nos 1 and 2, respectively. In the experiment that follows, the monoclonal IgM antibody 1A1 was raised specifically against the YML epitope using, as antigen/immunogen, a peptide having the sequence GGYMLGS (beta strand one with two flanking residues N and C terminal, SEQ ID No.2). This work is also described in WO 2010/099612, incorporated herein by reference.
[0193] General references: 263K hamster-adapted prions are described by Kimberlin et al., 1978. RML mouse-adapted prions are described by Chandler, R. L. (1961) (Lancet 1, 1378-1379). These may be used to infect mice or hamsters, using methods known in the art, for example those of Bueler H et al., 1993 (Cell 73:1339-1347), Oldstone et al., 2002, or Meade-White et al., 2009. Bolton et al., 1987 describe methods that may be used for isolation and purification of scrapie agent. Carlson et al., 1986 (Cell, 46:503-511) describes methods that may be used for clinical diagnosis of scrapie in mice and hamsters. Various transgenic mice overexpressing, partially expressing or lacking expression of PrP are described by Fischer et al., 1996 (EMBO J 15:1255-1264) and Weissmann et al., 2003 (British Medical Bulletin 66:43-60).
[0194] Brain tissues (normal and scrapie-infected mouse or hamster) were processed and analyzed as modified from Fischer et al 2000 (Nature 408:479-483). Briefly, 10% homogenates were made in PBS, 0.5% deoxycholate (Sigma), 0.5% NP-40. The total protein concentration in the homogenate was determined by BCA assay (Pierce) and adjusted to 5 mg/ml with homogenization buffer. For detection of PK-resistant material, 1.5 μl of homogenate was incubated with, or without, 0.15 μg PK (Sigma) at 37° C. for 60 minutes. Digestion was halted by addition of 20 mM PMSF (Sigma).
[0195] Antibodies were conjugated to magnetic beads and used for immunoprecipitation experiments as modified from Paramithiotis, 2003. Briefly, 7×108 magnetic beads (in 1 ml PBS) (Dynal; Lake Success, New York) were coupled according to the manufacturer's instructions to: 1A1, 8B4, 4E4 or IgM isotype control. Conjugated beads were washed and blocked according to the manufacturer's recommendations, then resuspended in 1 ml of PBS.
[0196] 10 μL of antibody-coupled beads were incubated with 1 μL of 10% brain homogenate in 6% detergent (3% Tween20 and 3% NP40 in PBS) for 3 hr at room temperature. Magnet-captured immune complexes were washed 3 times with 4% detergent (2% Tween20 and 2% NP40 in PBS), boiled in 4% SDS without reducing agents, and resolved on 15% Trisglycine or 4-12% bis-tris acrylamide gels (Invitrogen).
[0197] Immunoblotting was performed as described (Paramithiotis et al., 2003). Proteins were transferred onto PVDF membranes (Invitrogen). Membranes were blocked with 5% (w/v) dried non-fat milk. All incubations were done in TBST (25 mM Tris-HCl, 0.2 M NaCl, 0.5% Tween-20). Peroxidase activity was detected by chemiluminescence; enhanced ECL (Amersham) or superWest Dura (Pierce; Rockford, Ill.).
[0198] Immunoprecipitated samples were analyzed by Western blotting with 6D11-biotin as the primary antibody (1:5000) and Strep-HRP as the secondary antibody (1:5000). 8B4-bead acted as a positive control and was able to immunoprecipitate PrP from all the brain homogenate samples except the PrP Knocked Out mouse (K/O). Beads only, IgM-isotype-beads and 4E4-beads acted as negative controls and as expected, no PrP was immunoprecipitated, except two very faint bands in the RML and 263K lanes as immunoprecipitated by the 4E4. 1A1, an IgM antibody that was raised against the beta-1 strand of PrP, was able to immunoprecipitate scrapie prion proteins from both RML (mouse scrapie strain) and 263K (hamster scrapie strain). There is a faint band in the Tg20 lane possibly due to a small content of misfolded PrP in this Prnp overexpressing transgenic mouse brain. Data indicated that 1A1 was able to recognize only the scrapie PrP, but not the wild type PrP in both mouse and hamster brain.
Example 3
YML on the Surface of Tumors
[0199] The 1A1 antibody was tested for its ability to bind to both normal and tumor cells. As a control, the anti-PrPC antibody 6D11 was used to identify the level of expression of PrP on each cell type. The ability of antibodies to bind to cells was observed by flow cytometry using HUVEC (human umbilical vein endothelial cells) as the normal cell type. Eight types of tumor cells were tested. Five of the cancer cells tested are immortalized cell lines from mice (B16--melanoma, NSC34--motor neuron/neuroblastoma hybrid) and humans (HL60--promylocytic leukemia, MO3.13--oligodendrocyte/muscle hybrid, SiHa--cervical carcinoma). The remaining cancer cells tested are primary tumor cells that have been propagated by the Living Tumor Laboratory (LTL) at the British Columbia Cancer Agency. Using proprietary technology, primary human tumors are propagated under the kidney capsules of immunodeficient mice. This allows the original tumor architecture and phenotype to remain consistent with the originally harvested tumor. The three tumors that have been tested for binding to the 1A1 antibody are LTL-013 (large diffuse B-Cell lymphoma), LTL-257 (colorectal sarcoma) and LTL-323 (melanoma).
[0200] By flow cytometry, the 1A1 YML monoclonal antibody shows minimal binding to normal HUVEC cells compared to an isotype IgM control. 1A1 also shows no detectable binding to HL60 myeloid leukemia compared to IgM isotype control. HUVECs and HL60 cells have moderate and high levels, respectively, of 6D11 immunoreactivity, indicating that YML exposure is not a simple function of prion protein expression. The 1A1 antibody also shows detectable binding to seven other human and mouse tumor cell lines and LTL human tumors, all of which also display detectable cell surface 6D11 prion protein immunoreactivity.
[0201] To determine if an anti-DSE antibody can modify tumor progression in vivo, the 1A1 antibody was tested for its ability to modify growth of a murine melanoma tumor (B16) in female C57B1/6 mice. On day 0 of the study, 3×105 tumour cells were implanted subcutaneously into the flank of 12 mice. The mice were randomly assigned to two treatment groups. Group 1 was treated with PBS. Group 2 was treated with 1A1 antibody at 10 mg/kg. Mice were treated on days -1, 2 and 5.
[0202] Tumour growth was monitored by measuring tumour dimensions with calipers beginning on day 2. Tumour length and width measurements were obtained and tumour volumes were calculated according to the equation L×W2/2 with the length (mm) being the longer axis of the tumour. Mice were sacrificed once tumour burden was high, according to standard animal care procedures. There is a significant difference in tumour growth between the two groups (paired t-test=0.012; Wilcoxin=0.007), indicating that a therapeutic effect of the 1A1 antibody has occurred.
Example 4
Antibodies to the Rigid Loop Epitope of Misfolded Human PrP
[0203] Peptides comprising the sequence MDEYSNQNN (SEQ ID No. 9) were synthesized using standard methods and then coupled to carrier proteins. Prepared immunogens included both KLH-Cys-MEDYSNQNN and OVA-Cys-MDEYSNQNN. This work is described in the applicants co-pending U.S. Ser. No. 61/658,569 filed Jun. 12, 2012 and incorporated herein by reference.
[0204] New Zealand white rabbits were immunized subcutaneously with 0.4 mg peptide-KLH conjugates in complete Freund's adjuvant. After the initial immunization, animals were boosted several times every 2-3 weeks. The rabbit with the best titer in immunoassay was intravenously boosted with peptide antigen again, four days before the removal of the spleen. The hybridoma fusion was performed using conventional PEG cell fusion methodology. Splenocytes were harvested from the immunized rabbit and fused with rabbit plasmacytoma cells 240E-W2 (U.S. Pat. No. 5,675,063) using PEG4000 (Sigma Chemical, St. Louis, Mo.) and selected by HAT (hypoxanthine, aminopterin, and thymidine). At the end of selection hybridoma supernatants were collected and evaluated in various assays. Selected hybridomas were subsequently subcloned by limited dilution to obtain monoclonal hybridomas.
[0205] The antibody heavy and light chain genes for monoclonal ab120 were cloned from the hybridoma cells. Total RNA was extracted and reverse-transcribed to cDNA using the Qiagen TurboCapture mRNA kits. DNA fragments for L chain and the variable region (VH) of H chain of rabbit IgG were amplified by PCR with rabbit H and L chain primers. The L chain fragment was cloned into pTT5 mammalian expression vector and the VH fragment fused in-frame to the constant region of H chain pTT5 Heavy chain vector For each hybridoma clone, three plasmid DNA clones for H and L chains were sequenced and expressed as recombinant RabMAb for characterization.
[0206] Plasmids encoding the IgG heavy and light chains of ab120 were isolated from transformed E. coli using EndoFree® plasmid purification kit (Qiagen). Human HEK-293-6E cells were used for transient expression of ab120 antibody. The antibody plasmids were transfected into cells at logarithmic growth phase using FreeStyle® MAX Reagent 293 fectin (Invitrogen, Cat: 51-0031) and cultured in FreeStyle® 293 Expression Medium (Invitrogen, Cat: 12338-18) according to manufacturer's instructions. The transfected cells were grown at 37° C. with 5% CO2 in an orbital shaker for 7 days. The antibody secreted into the culture medium was collected by spinning at 7000 rpm for 15 minutes to remove cell debris. The cleared culture supernatant was purified by protein A chromatography (HiTrap® rProtein A FF, GE healthcare, CAT: 17-5080-01) under endotoxin free condition. Antibodies were eluted from the column in citrate elution buffer (SIGMA, CAT: C2404-100G) and adjusted to neutral pH with sodium bicarbonate buffer. The antibody preparation was concentrated and exchanged into PBS buffer. The concentration of IgG and endotoxin level in the final antibody preparation were determined by OD 280 nm quantitation and Tachypleus Amebocyte Lysate gel clot assay (Zhanjiang A&C Biological Ltd), respectively.
[0207] Monoclonal antibody ab120 was purified by protein A. Purified antibody was filter-sterilized and stored at 4 C in PBS buffer (pH 7.4). The protein concentration was determined by UV absorption 280 nm) assay and PBS buffer was used a blank buffer. The final concentration is the means from triplicate readings, and was given a QC requirement of >2 mg/ml.
[0208] To measure protein purity, SDS-PAGE was performed with Bio-Rad mini electrophoresis system according to the manufacturer's instructions. The gel was then stained with Coomassie brilliant blue. The resolving gel was 12% acrylamide and the stacking gel was 4% acrylamide, with sample loading at 4 ug/lane. The assayed sample showed 2 bands (Heavy chain and Light chain) in reduced SDS-PAGE, and one band (whole IgG molecule) in non-reduced SDS-PAGE.
[0209] Endotoxin level was also assessed by the Gel Clot Tachypleus Ameboycte Lysate (TAL) kit using endotoxin standards and endotoxin-free water. Results indicated an endotoxin level of <1 EU/ml protein.
[0210] Thus in a preferred embodiment, the antibody is provided as a preparation that exhibits (a) <about 1 EU/ml protein, (b) a concentration of greater than about 2 mg/ml, (c) and migration as a single protein band when measured by non-reducing SDS-PAGE at a loading dose of 4 ug/lane and detected at 280 nm.
[0211] Maxisorp 96-well plates were coated overnight at 2-8° C. with 100 ng/well of BSA-peptide in PBS. After blocking with PBST/casein, primary antibodies were added and incubated for 1 hour at room temperature. Rabbit antibodies were detected using goat anti-rabbit IgG-HRP and TMB substrate. After stopping the reaction with 0.25M sulfuric acid, absorbance was measured at 450 nm.
[0212] Recombinant PrP (Alicon) was mixed with LDS sample buffer (Life Technologies) and sample reducing agent (Life Technologies) and heated at 80° C. for 20 minutes. After cooling for 15 minutes, Maxisorp 96-well plates were coated with 100 ng/well of denatured PrP and incubated at 2-8° C. overnight. After blocking with PBST/BSA, primary antibodies were added and incubated for 1 hour at room temperature. Remaining steps were as described for anti-peptide ELISAs.
[0213] Maxisorp 96-well plates were coated overnight at 2-8° C. with 100 ng/well of goat anti-His-6 antibody (QED) in PBS. After blocking with PBST/BSA, His-PrP (Alicon) was added and incubated for 1 hour at room temperature. Addition of primary antibody and remaining steps were as described for anti-peptide ELISAs.
[0214] Adherent tumor cell lines and primary cells were detached from flasks using non-enzymatic cell-dissociation buffer (Invitrogen). Peripheral blood mononuclear cells were prepared from fresh citrated blood on the day of collection using standard Ficoll centrifugation methods. Other primary cells were frozen in 10% DMSO and thawed on the day of testing. Implanted tumors were surgically removed from mice. Tumors were chopped with scissors and then treated with collagenase/hyaluronidase (Worthington Biochemical) while shaking at 37° C. for 30 minutes. Individual tumor cells were collected by passing the mixture through a 40 μm screen.
[0215] Cells with Fc receptors were treated with 10% normal human serum to block the receptors. Cells were incubated with primary antibodies for 30 minutes at 2-8° C. Following washing, cells were incubated with goat anti-rabbit AF488 for 30 minutes at 2-8° C. After the final wash, cells were incubated in 1 μg/mL propidium iodide. Cells were analyzed using either a Becton Dickinson FACSCalibur or a Becton Dickinson FACS Canto II and FCS Express Software (DeNovo Systems).
[0216] The Octet QK system from ForteBio was used (by sub-contractor T-mab). Measurements were performed according to standard protocols. BSA-peptides were biotinylated and then coupled to a streptavidin-coated biosensor. The specific antibodies in the hybridoma supernatants were allowed to incubate with the treated biosensor.
[0217] Binding of antibodies to denatured PrP was performed by ELISA as described above. Binding of antibodies to tumor cells was performed by FACS as described above. Antibodies were titrated to provide binding curves. EC50 values were calculated using GraphPad software.
Results
[0218] The ProMis® algorithm (described in WO 2010/040209) was used to identify DSEs for human PrP. DSE3 is called the rigid loop epitope, and it is located between β-sheet 2 and α-helix 1.
[0219] Anti-DSE3 antibodies were developed using the specific sequence MDEYSNQNN, and two different immunogens, i.e., KLH-Cys-DSE3 and OVA-Cys-DSE3. Rabbits were immunized as described in the Materials and Methods and the antisera from the rabbits were evaluated. Rabbits made excellent responses to the immunogen peptides. In addition, antisera showed excellent binding to full length denatured PrP. After performing fusions, monoclonal antibodies were generated. Seven recombinant rabbit monoclonal antibodies raised against DSE3 were then fully evaluated.
[0220] Antibodies were tested for binding to the immunogen peptides, and all seven antibodies showed excellent titers. Kd values for peptide binding were determined using surface plasmon resonance. All antibodies showed very high affinity for peptides, with Kds in the 10-10M range.
[0221] All antibodies were then tested by ELISA for binding to denatured full-length recombinant PrP. One antibody exhibited a titer for denatured PrP that was similar to the anti-peptide affinities, in the 10-10 M range. Thus, the preferred antibody exhibits preferably an EC50 by this test that is at least better than 10-9M. The remaining antibodies showed logs lower affinity to denatured protein (10-6 to 10-9 M range) than to peptide. All antibodies were also tested by ELISA for binding to captured His-tagged PrP. None of the antibodies showed binding to captured His-PrP.
[0222] All seven antibodies were tested for binding to a panel of eleven tumor cell lines, six implanted primary human tumors, and nine normal cells. Only two antibodies showed binding to tumor cell lines (DSE3 ab90 and DSE3 ab120). When tested for binding to normal cells, DSE3 ab90 showed more binding to normal cells than to tumor cells. Although DSE3 ab120 also showed a small amount of binding to normal cells, this was less than the amount of binding observed against both tumor cell lines and passaged primary tumors. The binding of DSE ab120 was particularly strong against ovarian tumor cells, as the antibody bound well to five of six ovarian tumors tested, but did not bind to normal ovarian epithelial cells. PrP is expressed on all ovarian cells tested, although to varying degrees. In order to account for the differences in overall PrP levels, the binding of DSE3 ab120 was normalized to the binding of the control PrP antibody, 6H4.
[0223] For the six tumor cells, normalized DSE3 ab120 binding ranged from a low of 3.1 to a high of 71.6 (average=18.8), However, the normalized DSE3 ab120 binding was only 0.4 for the normal ovarian cells.
[0224] In order to determine the affinity of DSE3 ab120 to tumor cells, antibody titrations were performed on three ovarian tumor cell lines. Antibody titrations were also performed on two types of normal cells, and confirmed the earlier findings that DSE3 ab120 does not bind to these normal cells. Even though up to 40 ug/mL of antibody was tested, binding saturation was not reached on the tumor cells and affinities could not be determined. In the same experiments, the PrP control antibody 6H4 was also titrated and binding saturation was reached. For 6H4, the average calculated EC50 is 1.7×10-8M and there was no significant difference in the EC50 on tumor and normal cells. Since the binding of DSE3 ab120 to tumor cells is of lower affinity than 6H4, the EC50 for DSE3 ab120 must be lower than 1.7×10-8 M, and thus at least one log lower than the binding of DSE3 ab120 to denatured PrP (8.6×10-10 M).
[0225] The location of the CDRs within the antibody is determined by numbering amino acid residues with reference to the Kabat numbering system.
[0226] This antibody, herein designated ab120, thus displays an affinity for binding to misfolded PrP when present on ovarian cancer cells. Its ability to detect senescent cells is explored in the next Example.
Example 5
Senescent Cell Detection, PrP-Based
[0227] When primary cells, HUVECs are induced to undergo senescence, there is a proportion of cell surface PrP protein that stains for a misfolding-specific epitope referenced herein as the rigid loop.
[0228] More particularly, HUVEC cells were seeded at 50% confluence in 6-well culture plates and cultured in standard growth medium for 24 hours. Media was then removed and the cells were treated with 10 μg/ml Mitomycin C for 2 hrs in fresh cell culture medium. The solution was then removed and the cells were washed twice in PBS for about 30 seconds per wash. Lysosomal alkalinization was induced using the SA-β-gal staining kit, according to the supplier's instructions (Cell Biolabs, CBA-232). The cells were ultimately washed three times in PBS. A cell scraper was used to harvest the cells, and blocking with PBS containing 10% NGS for 30 minutes on ice. Those cells were then stained by first incubating the cells on ice with primary antibodies, i.e., antibody c-120 at 40 μg/ml and antibody 6D11 as control at 5 μg/ml, washing in cold PBS/2% NGS, and then incubating for 30 minutes on ice with secondary Alexa-488 labeled antibodies. Cells were then subjected to flow cytometry and analyzed. Results are shown in FIGS. 1 and 2.
[0229] When treated and untreated cells were incubated with either (1) a standard murine antibody (mIgG), (2) a PrP antibody that binds PrP in both natively and misfolded conformations (6D11), or (3) the c-120 PrP antibody that binds the rigid loop of PrP and thus binds selectively to PrP in a misfolded state, it was revealed (FIG. 2) that senescent HUVECs were bound selectively by the misfolded PrP antibody.
Example 6
Senescent Cell Detection, FasR-Based
[0230] The principles established herein are further demonstrated by targeting another and different cell surface protein first to determine if it, too, adopts a misfolded conformation when present on senescent cells, and then to determine if antibodies to that target can inhibit the growth of those target-positive cells.
[0231] Chosen as a second target was the protein FasR. Fas receptor (FasR) is known also as human tumour necrosis factor superfamily member 6 receptor (hTNFRSM6), and as CD95, and is implicated in cancer. It is a death receptor on the surface of of cells that leads to caspase-mediated programmed cell death (apoptosis). Antibodies to a misfolded form of this protein were prepared based on the epitopes predicted by Cashman et al, WO 2010/040209, as shown in Table 3:
TABLE-US-00010 TABLE 3 Human FASR (hTNFR6) 52-60 LHHDGQFCH 62 P25445 residues 1- 70-80 ARDCTVNGDEP 63 335 105-111 RLCDEGH 64 136-142 NSTVCEH 65 167-189 EEPSRSNLGWLCL 66
[0232] To raise antibodies against misfolded FasR, peptides comprising the FasR epitope sequences LHHDGQFCH (SEQ ID No. 62) and NSTVCEH (SEQ ID No. 65) were synthesized using standard methods and then coupled to carrier proteins. Prepared immunogens included both KLH-Cys-X and OVA-Cys-X, where X is NSTVCEH or LHHDGQFCH.
[0233] New Zealand white rabbits were immunized subcutaneously with 0.4 mg peptide-KLH conjugates in complete Freund's adjuvant. After the initial immunization, animals were boosted several times every 2-3 weeks. The rabbit with the best titer in immunoassay was intravenously boosted with peptide antigen again, four days before the removal of the spleen. The hybridoma fusion was performed using conventional PEG cell fusion methodology. Splenocytes were harvested from the immunized rabbit and fused with rabbit plasmacytoma cells 240E-W2 (U.S. Pat. No. 5,675,063) using PEG4000 (Sigma Chemical, St. Louis, Mo.) and selected by HAT (hypoxanthine, aminopterin, and thymidine). At the end of selection hybridoma supernatants were collected and evaluated in various assays. Selected hybridomas were subsequently subcloned by limited dilution to obtain monoclonal hybridomas.
[0234] The antibody heavy and light chain genes for monoclonal AMF-3a-118 and AMF-3d-19 were cloned from the hybridoma cells. Total RNA was extracted and reverse-transcribed to cDNA using the Qiagen TurboCapture mRNA kits. DNA fragments for L chain and the variable region (VH) of H chain of rabbit IgG were amplified by PCR with rabbit H and L chain primers. The L chain fragment was cloned into pTT5 mammalian expression vector and the VH fragment fused in-frame to the constant region of H chain pTT5 Heavy chain vector For each hybridoma clone, three plasmid DNA clones for H and L chains were sequenced and expressed as recombinant RabMAb for characterization.
[0235] Plamids encoding the IgG heavy and light chains of AMF-3a-118 and AMF-3d-19 were isolated from transformed E. coli using EndoFree® plasmid purification kit (Qiagen). Human HEK-293-6E cells were used for transient expression of AMF-3a-118 and AMF-3d-19 antibodies. The antibody plasmids were transfected into cells at logarithmic growth phase using FreeStyle® MAX Reagent 293 fectin (Invitrogen, Cat: 51-0031) and cultured in FreeStyle® 293 Expression Medium (Invitrogen, Cat: 12338-18) according to manufacturer's instructions. The antibody secreted into the culture medium was collected by spinning at 7000 rpm for 15 minutes to remove cell debris. The cleared culture supernatant was purified by protein A chromatography (HiTrap® rProtein A FF, GE healthcare, CAT: 17-5080-01) under endotoxin free condition. Antibodies were eluted from the column in citrate elution buffer (SIGMA, CAT: C2404-100G) and adjusted to neutral pH with sodium bicarbonate buffer. The antibody preparations were concentrated and exchanged into PBS buffer.
[0236] Purified antibody was filter-sterilized and stored at 4° C. in PBS buffer (pH 7.4). The protein concentration was determined by UV absorption 280 nm) assay and PBS buffer was used a blank buffer. To measure protein purity, SDS-PAGE was performed with Bio-Rad mini electrophoresis system according to the manufacturer's instructions. The gel was then stained with Coomassie brilliant blue. The resolving gel was 12% acrylamide and the stacking gel was 4% acrylamide. The assayed samples showed 2 bands (Heavy chain and Light chain) in reduced SDS-PAGE, and one band (whole IgG molecule) in non-reduced SDS-PAGE.
[0237] Thus in a preferred embodiment, the antibody is provided as a preparation that exhibits (a) a concentration of greater than about 1 mg/ml, (b) and migration as a single protein band when measured by non-reducing SDS-PAGE.
[0238] Maxisorp 96-well plates were coated overnight at 2-8° C. with 100 ng/well of BSA-peptide in PBS. After blocking with PBST/casein, primary antibodies were added and incubated for 1 hour at room temperature. Rabbit antibodies were detected using goat anti-rabbit IgG-HRP and TMB substrate. After stopping the reaction with 0.25M sulfuric acid, absorbance was measured at 450 nm.
[0239] Recombinant Fas extracellular domain-Fc fusion protein (Aragen) was mixed with LDS sample buffer (Life Technologies) and sample reducing agent (Life Technologies) and heated at 80° C. for 20 minutes. After cooling for 15 minutes, Maxisorp 96-well plates were coated with 100 ng/well of denatured Fas and incubated at 2-8° C. overnight. After blocking with PBST/BSA, primary antibodies were added and incubated for 1 hour at room temperature. Remaining steps were as described for anti-peptide ELISAs.
[0240] His-tagged Fas (Creative Biomart) was mixed with Talon Dynabeads (Invitrogen) in round-bottom polystyrene 96-well plates for 30 minutes at room temperature. After beads were collected on a magnet, beads were washed 3× with PBST, then mixed with primary antibodies at room temperature for 60 minutes. Bound primary antibodies were detected with goat anti-rabbit IgG-HRP and TMB substrate. Supernatant was transferred to a flat-bottom 96-well plate (Nunc), the reaction stopped with 0.25M sulfuric acid, and absorbance was measured at 450 nm.
[0241] Antibodies having particularly good binding profiles include antibody AMF-3a-118 for the peptide LHHDGQFCH, and antibody AMF-3d-19 for the peptide NSTVCEH. These antibodies have the full length protein sequences set out in SEQ ID No. 91 (light chain) and SEQ ID No.89 (heavy chain) for 3a-118, and sequences set out in SEQ ID No. 105 (light chain) and SEQ ID No.103 (heavy chain) for 3d-19. Polynucleotides encoding them have the sequences set out in SEQ ID No. 99 (heavy chain) and SEQ ID No.101 (light chain) for 3a-118 and SEQ ID No. 113 (heavy chain) and SEQ ID No. 115 (light chain) for antibody 3d-19. The important complementarity determining regions (CDRs) of these antibodies are set out below:
AMF-3a-118
[0242] For the heavy chain:
TABLE-US-00011 CDR1 (SEQ ID No. 93) CDR2 (SEQ ID No. 94) CDR3 (SEQ ID No. 95)
For the light chain:
TABLE-US-00012 CDR1 (SEQ ID No. 96) CDR2 (SEQ ID No. 97) CDR3 (SEQ ID No. 98)
AMF-3d-19
[0243] For the heavy chain:
TABLE-US-00013 CDR1 (SEQ ID No. 107) CDR2 (SEQ ID No. 108) CDR3 (SEQ ID No. 109)
For the light chain:
TABLE-US-00014 CDR1 (SEQ ID No. 110) CDR2 (SEQ ID No. 111) CDR3 (SEQ ID No. 112)
[0244] These misfolded FasR antibodies were assessed for their ability to detect senescent cells, using the approach already described above. More particularly, HUVEC cells were seeded at 50% confluence in 6-well culture plates and cultured in standard growth medium for 24 hours. Media was then removed and the cells were treated with 10 μg/ml Mitomycin C for 2 hrs in fresh cell culture medium. The solution was then removed and the cells were washed twice in PBS for about 30 seconds per wash. Lysosomal alkalinization was induced using the SA-β-gal staining kit, according to the supplier's instructions (Cell Biolabs, CBA-232). The cells were ultimately washed three times in PBS. A cell scraper was used to harvest the cells, and blocking with PBS containing 10% NGS for 30 minutes on ice. Those cells were then stained by first incubating the cells on ice with primary antibodies, i.e., antibodies 3a-118 and 3d-19 at 40 μg/ml and antibody 6D11 as control at 5 μg/ml, washing in cold PBS/2% NGS, and then incubating for 30 minutes on ice with secondary Alexa-488 labeled antibodies. Cells were then subjected to flow cytometry and analyzed. Results are shown in FIGS. 3 and 4.
[0245] When treated and untreated cells were incubated with either (1) a standard murine antibody (mIgG), (2) a PrP antibody that binds PrP in both natively and misfolded conformations (6D11), or (3) the FasR antibodies that binds the noted exposed epitopes and thus bind selectively to FasR in a misfolded state, it was revealed (FIGS. 3 and 4) that senescent HUVECs were bound selectively by the misfolded FasR antibodies.
[0246] All citations are herein incorporated by reference, as if each individual publication was specifically and individually indicated to be incorporated by reference herein and as though it were fully set forth herein.
TABLE-US-00015 Table of Sequences SEQ ID Subject Sequence 1 Misfolded PrP epitope LGGYML 2 Misfolded PrP epitope GGYMLGS 3 Misfolded PrP epitope HFGSDYED 4 Misfolded PrP epitope SDYED 5 Misfolded PrP epitope RYYRENMH 6 Misfolded PrP epitope RENMH 7 Misfolded PrP epitope QVYYRPM 8 Misfolded PrP epitope PMDEYSNQNN 9 Misfolded PrP epitope MDEYSNQNN 10 Misfolded PrP epitope KQHTVTTTTKGEN 11 Misfolded PrP epitope ARDCTVNGDEP 12 Misfolded PrP epitope RLCDEGH 13 Misfolded PrP epitope NSTVCEH 14 Misfolded PrP epitope EEPSRSNLGWLCL 15 Misfolded PrP sequence RYYRE 16 Misfolded PrP sequence RYYREN 17 Misfolded PrP sequence RYYRENM 18 Misfolded PrP sequence DRYYRENMH 19 Misfolded PrP sequence DRYYRENM 20 Misfolded PrP sequence VYYRPM 21 Misfolded PrP sequence QVYYRP 22 Misfolded PrP sequence QVYYR 23 Misfolded PrP sequence QVYYRPMD 24 Misfolded PrP sequence QVYYRPM 25 Misfolded PrP sequence GGYMLG 26 Misfolded PrP sequence GYMLGS 27 Misfolded PrP sequence GGYML 28 Misfolded PrP sequence YMLGS 29 Misfolded PrP sequence GYML 30 Misfolded PrP sequence YMLG 31 Misfolded PrP sequence LGGYML 32 Misfolded PrP sequence LGGYMLG 33 Misfolded PrP sequence YML 34 Misfolded PrP sequence PMDEYSNQNN 35 Misfolded PrP sequence DEYSNQNN 36 Misfolded PrP sequence MDEYSNQ 37 Ab c-120 light chain MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQSSQSLYNKNWLSWYQKKPGQPPKLL IYKASTLESGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSADCTAFGGGTEVVVKGDPVAPT VLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQY NSHKEYTCKVTQGTTSVVQSFNRGDC 38 Ab c-120 heavy chain METGLRWLLLVAVLKGVQCQSVEESGGHLVTPGTPLTLTCTVSGIDLSTYAMGWVRQAPGKGLEWIGVITK SGNTYYASWAKGRFAISKTSTTVDLKITSPTTEDTATYFCGRYGIGVSYYDIWGPGTLVTVSSGQPKAPSV FPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVT CNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQ FTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLE PKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSE WQRGDVFTCSVMHEALHNHYTQKSISRSPGK 39 Ab c-120 light chain variable region (VL) MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQSSQSLYNKNWLSWYQKKPGQPPKLL IYKASTLESGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSADCTAFGGGTEVVV 40 Ab c-120 heavy chain variable region (VH) METGLRWLLLVAVLKGVQCQSVEESGGHLVTPGTPLTLTCTVSGIDLSTYAMGWVRQAPGKGLEWIGVITK SGNTYYASWAKGRFAISKTSTTVDLKITSPTTEDTATYFCGRYGIGVSYYDIWGPGTLVTVSSGQ 41 Ab c-120 CDR1 heavy TYAMG 42 Ab c-120 CDR2 heavy VITKSGNTYYASWAKG 43 Ab c-120 CDR3 heavy YGIGVSYYDI 44 Ab c-120 CDR1 light QSSQSLYNKNWLS 45 Ab c-120 CDR2 light KASTLES 46 Ab c-120 CDR3 light QGEFSCSSADCTA 47 misfolded CD44 epitope NGRYSIS 48 misfolded CD44 epitope EGHVV 49 misfolded CD44 epitope TSNTSNYDT 50 misfolded CD44 epitope NRDGTRYVQKG 51 misfolded CD44 epitope ANNTFVYILTSNTSNYDT 52 misfolded hTNFR epitope IHPQNNSICCT 53 misfolded hTNFR epitope NDCPGPGQDTD 54 misfoided hTNFR epitope YWSENLF 55 misfoided hTNFR epitope HLSCQEKQN 56 misfolded hTNFR epitope CTCHAGFFLRENECV 57 misfolded hNOTCH1 epitope CEHAGKC 58 misfolded hNOTCH1 epitope MPGYEGVHC 59 misfolded hNOTCH1 epitope CLHNGRC 60 misfolded hNOTCH1 epitope KINEF 61 misfolded hNOTCH1 epitope ECASSPCLHNGRCLDKINEFQCECP 62 misfolded hFas epitope LHHDGQFCH 63 misfolded hFas epitope ARDCTVNGDEP 64 misfolded hFas epitope RLCDEGH 65 misfolded hFas epitope NSTVCEH 66 misfolded hFas epitope EEPSRSNLGWLCL 67 misfolded hEGFR epitope SNKLTQLFTFEDHFL 68 misfolded hEGFR epitope VQRNYDLSFLK 69 misfolded hEGFR epitope IRGNMYYENSYAL 70 misfolded hEGFR epitope VLSNYDANKTG 71 misfolded hEGFR epitope IVSSDFLSNMSMD 72 misfolded hEGFR epitope FLSNMSMDFQNHLGS 73 misfolded hEGFR epitope WGAGEE 74 misfolded hEGFR epitope PPLMLYNPTTYQMDVNPE 75 misfolded hEGFR epitope PEGKYSFGAT 76 misfolded hEGFR epitope RNYVVTDHGS 77 misfolded hEGFR epitope GADSYEMEEDGVRK 78 misfolded hEGFR epitope NGIGIGEFKDSLSIN 79 misfolded hEGFR epitope ATNIKHFKN 80 misfolded hEGFR epitope LPVAFRGDSFTHTPPL 81 misfolded hEGFR epitope KIISNRGENS 82 misfolded CD38 epitope YTEIHPEMRHVDCQS 83 misfolded CD38 epitope GEFATSKIN 84 misfolded CD38 epitope WRKDCSN 85 misfolded CD38 epitope GSRSKIFDKDS 86 misfolded CD38 epitope IHGGREDSRDL 87 DNA for ab c-120 heavy chain ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGT- CCGG GGGTCACCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTACCTAT- GCAA TGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTAAAAGTGGTAACACATA- CTAC GCGAGCTGGGCGAAAGGCCGATTCGCCATCTCCAAAACCTCGACCACGGTGGATCTAAAGATCACCAGTCCGA- CAAC CGAGGACACGGCCACCTATTTCTGTGGCAGATATGGTATTGGTGTTTCTTACTATGACATCTGGGGCCCAGGC- ACTC TGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACC- GAGC TCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCA- CCCT CACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGC- GTGA CCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGC- GCCC TCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAA- AACC CAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCC- GAGG TGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAA- CAGC ACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAG- TCCA CAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTC- TACA CCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCC- TTCC GACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACA- GCGA CGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGC- TCCG TGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 88 DNA coding for c-120 light chain ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAG- TGCT GACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGT- CTTT ATAATAAGAACTGGTTATCCTGGTATCAGAAGAAACCAGGGCAGCCTCCTAAGCTCCTGATCTACAAGGCATC- CACT CTGGAATCTGCGGTCTCATCGCGGTTCAAGGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCG- TGCA GTGTGACGATGCTGCCACTTACTACTGTCAAGGCGAATTTAGTTGTAGTAGTGCTGATTGTACGGCTTTCGGC- GGAG GGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGT- GGCA ACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATG- GCAC CACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGC- ACTC TGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGT- CGTC CAGAGCTTCAATAGGGGTGACTGTTAG 89 AMF-3a-118 heavy chain M E T G L R W L L L V A V L K G V Q C Q S V E E S G G R L V T P G T P L T L T C K A S G F S L S D S R V S W V R Q A P G K G L E W I G I V G I G W N I Y H A N W A K G R F T I S K T S S T T V D L K I T S P T V E D T A T Y F C A R G L G G G T V I W G P G T L V T V S L G Q P K A P S V F P L A P C C G D T P S S T V T L G C L V K G Y L P E P V T V T W N S G T L T N G V R T F P S V R Q S S G L Y S L S S V V S V T S S S Q P V T C N V A H P A T N T K V D K T V A P S T C S K P T C P P P E L L G G P S V F I F P P K P K D T L M I S R T P E V T C V V V D V S Q D D P E V Q F T W Y I N N E Q V R T A R P P L R E Q Q F N S T I R V V S T L P I A H Q D W L R G K E F K C K V H N K A L P A P I E K T I S K A R G Q P L E P K V Y T M G P P R E E L S S R S V S L T C M I N G F Y P S D I S V E W E K N G K A E D N Y K T T P A V L D S D G S Y F L Y S K L S V P T S E W Q R G D V F T C S V M H E A L H N H Y T Q K S I S R S P G K -- 90 AMF-3a-118 heavy chain variable region M E T G L R W L L L V A V L K G V Q C Q S V E E S G G R L V T P G T P L T L T C K A S G E S L S D S R V S W V R Q A P G K G L E W I G I V G I G W N I Y H A N W A K G R F T I S K T S S T T V D L K I T S P T V E D T A T Y F C A R G L G G G T V I W G P G T L V T V S L 91 AMF-3a-118 light chain M D T R A P T Q L L G L L L L W L P G A T F A Q V L T Q T P A S V S A A V G G T V T I S C Q S S E S V Y K N N Y L S W F Q Q K P G Q P P K L L I Y E A S K L A S G V S T R F K G S G S G T Q F T L T I S G V Q C D D A A T Y Y C L G E F S C Y S G D C G T F G G G T A V V V K G D P V A P T V L I F P P A A D Q V A T G T V T I V C V A N K Y F P D V T V T W E V D G T T Q T T G I E N S K T P Q N S A D C T Y N L S S T L T L T S T Q Y N S H K E Y T C K V T Q G T T S V V Q S F N R G D C -- 92 AMF-3a-118 light chain variable region M D T R A P T Q L L G L L L L W L P G A T F A Q V L T Q T P A S V S A A V G G T V T I S C Q S S E S V Y K N N Y L S W F Q Q K P G Q P P K L L I Y E A S K L A S G V S T R F K G S G S G T Q F T L T I S G V Q C D D A A T Y Y C L G E F S C Y S G D C G T F G G G T A V V V K 93 Ab 3a-118 CDR1 heavy DSRVS 94 Ab 3a-118 CDR2 heavy IVGIGWNIYHANWAKG 95 Ab 3a-118 CDR3 heavy GLGGGTVI 96 Ab 3a-118 CDR1 light QSSESVKNNYLS 97 Ab 3a-118 CDR2 light EASKLAS 98 Ab 3a-118 CDR3 light LGEFSCYSGDCGT 99 AMF-3a-118 heavy chain-encoding DNA ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGT- CCGG GGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCT- AGAG TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATA- CCAC GCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTC- CGAC AGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACC-
CTGG TCACCGTCTCCTTA GGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCC- TGGG CTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTA- CGCA CCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCA- GCCC GTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCA- AGCC CACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTC- ATGA TCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATG- GTAC ATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGG- TCAG CACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTC- CCGG CCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCC- CCGG GAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGG- AGTG GGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTC- CTCT ACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGC- CTTG CACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 100 AMF-3a-118 heavy chain variable region-encoding DNA ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGT- CCGG GGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCT- AGAG TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATA- CCAC GCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTC- CGAC AGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACC- CTGG TCACCGTCTCCTTA 101 AMF 3a-118 light chain-encoding DNA ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAG- TGCT GACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTGAGAGT- GTTT ATAAGAACAACTACTTATCCTGGTTTCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACGAAGCATC- CAAA CTGGCATCTGGGGTCTCAACGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCG- TGCA GTGTGACGATGCTGCCACATACTACTGTCTAGGCGAATTTAGTTGTTATAGTGGTGATTGTGGTACTTTCGGC- GGAG GGACCGCGGTGGTGGTCAAA GCTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTCATCAGGTGGCAACTGCAACAGTCACCA- TCGT GTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGC- ATCG AGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCAC- ACAG TACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGG- GTGA CTGTTAG 102 AMF 3a-118 light chain variable region-encoding DNA ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAG- TGCT GACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTGAGAGT- GTTT ATAAGAACAACTACTTATCCTGGTTTCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACGAAGCATC- CAAA CTGGCATCTGGGGTCTCAACGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCG- TGCA GTGTGACGATGCTGCCACATACTACTGTCTAGGCGAATTTAGTTGTTATAGTGGTGATTGTGGTACTTTCGGC- GGAG GGACCGCGGTGGTGGTCAAA 103 AMF3d-19 heavy chain M E T G L R W L L L V A V L K G V Q C Q S L E E S G G R L V T P G T P L T L T C T V S G F S L S R N A I N W V R Q A P G K G L E Y I G I I G S S G V T Y Y A S W A K G R F T I S R T S T T V D L K I T S P T T E D T A T Y F C A R N L Y T G G S N D N L W G P G T L V T V S S G Q P K A P S V F P L A P C C G D T P S S T V T L G C L V K G Y L P E P V T V T W N S G T L T N G V R T F P S V R Q S S G L Y S L S S V V S V T S S S Q P V T C N V A H P A T N T K V D K T V A P S T C S K P T C P P P E L L G G P S V F I F P P K P K D T L M I S R T P E V T C V V V D V S Q D D P E V Q F T W Y I N N E Q V R T A R P P L R E Q Q F N S T I R V V S T L P I A H Q D W L R G K E F K C K V H N K A L P A P I E K T I S K A R G Q P L E P K V Y T M G P P R E E L S S R S V S L T C M I N G F Y P S D I S V E W E K N G K A E D N Y K T T P A V L D S D G S Y F L Y S K L S V P T S E W Q R G D V F T C S V M H E A L H N H Y T Q K S I S R S P G K -- 104 AMF3d-19 heavy chain variable region M E T G L R W L L L V A V L K G V Q C Q S L E E S G G R L V T P G T P L T L T C T V S G F S L S R N A I N W V R Q A P G K G L E Y I G I I G S S G V T Y Y A S W A K G R F T I S R T S T T V D L K I T S P T T E D T A T Y F C A R N L Y T G G S N D N L W G P G T L V T V S S 105 AMF 3d-19 light chain M D T R V P T Q L L G L L L L W L P G A T F A Q V L T Q T P S P V S A A V G G T V T I N C Q A S K S V y N N V Q L S W F Q Q K P G Q P P K R L I Y Y A S T L A S G V P S R F K G S G S G T Q F T L T I S D V Q C D D V A T Y Y C A G G Y S S S S D N A F G G G T E V V V K G D P V A P T V L I F P P A A D Q V A T G T V T I V C V A N K Y F P D V T V T W E V D G T T Q T T G I E N S K T P Q N S A D C T Y N L S S T L T L T S T Q Y N S H K E y T C K V T Q G T T S V V Q S F N R G D C -- 106 AMF 3d-19 light chain variable region M D T R V P T Q L L G L L L L W L P G A T F A Q V L T Q T P S P V S A A V G G T V T I N C Q A S K S V Y N N V Q L S W F Q Q K P G Q P P K R L I Y Y A S T L A S G V P S R F K G S G S G T Q F T L T I S D V Q C D D V A T Y Y C A G G Y S S S S D N A F G G G T E V V V K 107 Ab 3d-19 CDR1 heavy RNAIN 108 Ab 3d-19 CDR2 heavy ITGSSGVTYYASWAKG 109 Ab 3d-19 CDR3 heavy NLYTGGSNDNL 110 Ab 3d-19 CDR1 light QASKSVYNNVQLS 111 Ab 3d-19 CDR2 light YASTLAS 112 Ab 3d-19 CDR3 light AGGYSSSSDNA 113 AMF 3d-19 heavy chain-encoding DNA ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGTTGGAGGAGT- CCGG GGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTCGCAAT- GCAA TAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTGGTAGTAGTGGTGTCACATA- CTAC GCGAGCTGGGCAAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGA- CAAC CGAGGACACGGCCACCTATTTTTGTGCCAGAAATCTTTATACTGGTGGTAGTAATGATAACTTGTGGGGCCCA- GGCA CCCTGGTCACCGTCTCCTCA GGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCC- TGGG CTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTA- CGCA CCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCA- GCCC GTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCA- AGCC CACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTC- ATGA TCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATG- GTAC ATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGG- TCAG CACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTC- CCGG CCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCC- CCGG GAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGG- AGTG GGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTC- CTCT ACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGC- CTTG CACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 114 AMF 3d-19 heavy chain variable region-encoding DNA ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGTTGGAGGAGT- CCGG GGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTCGCAAT- GCAA TAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTGGTAGTAGTGGTGTCACATA- CTAC GCCAGCTGGGCAAAAGCCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGA- CAAC CGAGGACACGGCCACCTATTTTTGTGCCAGAAATCTTTATACTGGTGGTAGTAATGATAACTTGTGGGGCCCA- GGCA CCCTGGTCACCGTCTCCTCA 115 AMF 3d-19 light chain-encoding DNA ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGT- CCGG GGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCT- AGAG TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATA- CCAC GCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTC- CGAC AGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACC- CTGC TCACCGTCTCCTTA GGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCC- TGGG CTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTA- CGCA CCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCA- GCCC GTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCA- AGCC CACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTC- ATGA TCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATG- GTAC ATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGG- TCAG CACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTC- CCGG CCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCC- CCGG GAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGG- AGTG GGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTC- CTCT ACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGC- CTTG CACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 116 AMF 3d-19 light chain variable region-encoding DNA ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGCTGTCCAGTGTCAGTCGGTGGAGGACT- CCGG GGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCT- AGAG TGAGCTGGGTCCGCCAGGCTCCAGGGAACCGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATA- CCAC GCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTC- CGAC AGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACC- CTGG TCACCGTCTCCTTA 119 DNA for ab c-120 heavy chain (lower case = non-coding restriction site) aagcttgtacccttcacc ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGT- CCGG GGGTCACCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTACCTAT- GCAA TGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTAAAAGTGGTAACACATA- CTAC
GCGAGCTGGGCGAAAGGCCGATTCGCCATCTCCAAAACCTCGACCACCGTGGATCTAAAGATCACCAGTCCGA- CAAC CGAGGACACGGCCACCTATTTCTGTGGCAGATATGGTATTGGTGTTTCTTACTATGACATCTGGGGCCCAGGC- ACTC TGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTCCTGCGGGGACACACC- CAGC TCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCA- CCCT CACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGC- GTGA CCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGC- GCCC TCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAA- AACC CAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCC- GAGG TGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAA- CAGC ACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAG- TCCA CAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTC- TACA CCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCC- TTCC GACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACA- GCGA CGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGC- TCCG TGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA gcgctgtgccggcgagctgcggccgc 120 DNA coding for c-120 light chain (lower case = non-coding restriction site) aagcttgtacccttcacc ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAG- TGCT GACCCAGACTCCATCCCCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGT- CTTT ATAATAAGAACTGGTTATCCTGGTATCAGAAGAAACCAGGGCAGCCTCCTAAGCTCCTGATCTACAAGGCATC- CACT CTGGAATCTGGGGTCTCATCGCGGTTCAAGGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCG- TGCA GTGTGACGATGCTGCCACTTACTACTGTCAAGGCGAATTTAGTTGTAGTAGTGCTGATTGTACGGCTTTCGGC- GGAG GGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGT- GGCA ACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATG- GCAC CACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGC- ACTC TGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGT- CGTC CAGAGCTTCAATAGGGGTGACTGTTAG agtgagagcggccgc 121 AMF-3a-118 heavy chain-encoding DNA (sequence before ATG and after TGA, vector sequence; underlined sequence restriction site) AAGCTTGTACCCTTCACC ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGT- CCGG GGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCT- AGAG TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCGTTGGCATTGGTTGGAATATATA- CCAC GCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTC- CGAC AGTCGAGGACACGGCCACCTATTTCTGTGCCAGAGGTCTGGGTGGTGGTACTGTCATCTGGGGCCCAGGCACC- CTGG TCACCGTCTCCTTA GGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCC- TGGG CTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTA- CGCA CCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGGAGCGTGGTGAGCGTGACCTCAAGCAGCCA- GCCC GTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCA- AGCC CACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTC- ATGA TCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATG- GTAC ATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGG- TCAG CACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTC- CCGG CCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCC- CCGG GAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGG- AGTG GGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTC- CTCT ACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGC- CTTG CACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA GCGCTGTGCCGGCGAGCTGCGGCCGC 122 AMF 3a-118 light chain-encoding DNA (sequence before ATG and after TAG, vector sequence; underlined sequence restriction site) aagcttgtacccttcacc ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAG- TGCT GACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTGAGAGT- GTTT ATAAGAACAACTACTTATCCTGGTTTCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACGAAGCATC- CAAA CTGGCATCTGGGGTCTCAACGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCG- TGCA GTGTGACGATGCTGCCACATACTACTGTCTAGGCGAATTTAGTTGTTATAGTGGTGATTGTGGTACTTTCGGC- GGAG GGACCGCGGTGGTGGTCAAA GGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCA- TCGT GTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGC- ATCG AGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCAC- ACAG TACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGG- GTGA CTGTTAG Agtgagagcggccgc 123 AMF 3d-19 heavy chain-encoding DNA (sequence before ATG and after TGA, vector sequence; underlined sequence restriction site) aagcttgtacccttcacc ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGTTGGAGGAGT- CCGG GGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTCGCAAT- GCAA TAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTGGTAGTAGTGGTGTCACATA- CTAC GCGAGCTGGGCAAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGA- CAAC CGAGGACACGGCCACCTATTTTTGTGCCAGAAATCTTTATACTGGTGGTAGTAATGATAACTTGTGGGGCCCA- GGCA CCCTGGTCACCGTCTCCTCA GGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCC- TGGG CTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTA- CGCA CCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTCGTGAGCGTGACCTCAAGCAGCCA- GCCC GTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTCGACAAGACCGTTGCGCCCTCGACATGCAGCA- AGCC CACGTGCCCACCCCCTGAACTCCTGGCGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAACGACACCCTC- ATGA TCTCACCCACCCCCGAGCTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCACTTCACATG- GTAC ATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGG- TCAG CACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTC- CCGG CCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCC- CCGG GAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGG- AGTG CGAGAAGAACCGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTC- CTCT ACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGC- CTTG CACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA Gcgctgtgccggcgagctgcggccgc 124 AMF 3d-19 light chain-encoding DNA (sequence before ATG and after TGA, vector sequence; underlined sequence restriction site) aagcttgtacccttcacc ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCCCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGT- CCGG GGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTGACTCT- AGAG TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATCGATCCGAATCGTTGGCATTGGTTGGAATATATA- CCAC GCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACGTCGTCGACCACGGTGGATTTGAAAATCACCAGTC- CGAC AGTCGAGGACACGGCCACCTATTTCTGTGCCAGACGTCTGGGTGGTGCTACTGTCATCTGGGGCCCAGGCACC- CTGG TCACCGTCTCCTTA GGGCAACCTAACGCTCCATCAGTCTTCCCACTGCCCCCCTGCTGCGGCGACACACCCAGCTCCACGGTGACCC- TGGG CTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTA- CGCA CCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCA- GCCC GTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCA- AGCC CACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTC- ATGA TCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATG- GTAC ATAAACAACGAGCAGGTGCCCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGG- TCAG CACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTC- CCGG CCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCC- CCGG GAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGG- AGTG GGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTC- CTCT ACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGC- CTTG CACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA gcgctgtgccggcgagctgcggccgc
Sequence CWU
1
1
12416PRTHomo Sapien 1Leu Gly Gly Tyr Met Leu 1 5
27PRTHomo Sapien 2Gly Gly Tyr Met Leu Gly Ser 1 5
38PRTHomo Sapien 3His Phe Gly Ser Asp Tyr Glu Asp 1 5
45PRTHomo Sapien 4Ser Asp Tyr Glu Asp 1 5
58PRTHomo Sapien 5Arg Tyr Tyr Arg Glu Asn Met His 1 5
65PRTHomo Sapien 6Arg Glu Asn Met His 1 5
77PRTHomo Sapien 7Gln Val Tyr Tyr Arg Pro Met 1 5
810PRTHomo Sapien 8Pro Met Asp Glu Tyr Ser Asn Gln Asn Asn 1
5 10 99PRTHomo Sapien 9Met Asp Glu Tyr Ser Asn Gln
Asn Asn 1 5 1013PRTHomo Sapien 10Lys Gln
His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn 1 5
10 1111PRTHomo Sapien 11Ala Arg Asp Cys Thr Val Asn
Gly Asp Glu Pro 1 5 10 127PRTHomo
Sapien 12Arg Leu Cys Asp Glu Gly His 1 5
137PRTHomo Sapien 13Asn Ser Thr Val Cys Glu His 1 5
1413PRTHomo Sapien 14Glu Glu Pro Ser Arg Ser Asn Leu Gly Trp Leu Cys
Leu 1 5 10 155PRTHomo Sapien
15Arg Tyr Tyr Arg Glu 1 5 166PRTHomo Sapien 16Arg Tyr Tyr
Arg Glu Asn 1 5 177PRTHomo Sapien 17Arg Tyr Tyr Arg
Glu Asn Met 1 5 189PRTHomo Sapien 18Asp Arg Tyr
Tyr Arg Glu Asn Met His 1 5 198PRTHomo
Sapien 19Asp Arg Tyr Tyr Arg Glu Asn Met 1 5
206PRTHomo Sapien 20Val Tyr Tyr Arg Pro Met 1 5
216PRTHomo Sapien 21Gln Val Tyr Tyr Arg Pro 1 5
225PRTHomo Sapien 22Gln Val Tyr Tyr Arg 1 5 238PRTHomo
Sapien 23Gln Val Tyr Tyr Arg Pro Met Asp 1 5
247PRTHomo Sapien 24Gln Val Tyr Tyr Arg Pro Met 1 5
256PRTHomo Sapien 25Gly Gly Tyr Met Leu Gly 1 5
266PRTHomo Sapien 26Gly Tyr Met Leu Gly Ser 1 5
275PRTHomo Sapien 27Gly Gly Tyr Met Leu 1 5 285PRTHomo
Sapien 28Tyr Met Leu Gly Ser 1 5 294PRTHomo Sapien 29Gly
Tyr Met Leu 1 304PRTHomo Sapien 30Tyr Met Leu Gly 1
316PRTHomo Sapien 31Leu Gly Gly Tyr Met Leu 1 5
327PRTHomo Sapien 32Leu Gly Gly Tyr Met Leu Gly 1 5
333PRTHomo Sapien 33Tyr Met Leu 1 3410PRTHomo Sapien 34Pro
Met Asp Glu Tyr Ser Asn Gln Asn Asn 1 5
10 358PRTHomo Sapien 35Asp Glu Tyr Ser Asn Gln Asn Asn 1 5
367PRTHomo Sapien 36Met Asp Glu Tyr Ser Asn Gln 1
5 37239PRTOryctolagus cuniculus 37Met Asp Thr Arg Ala Pro
Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr
Gln Thr Pro Ser Pro 20 25
30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser
Ser 35 40 45 Gln
Ser Leu Tyr Asn Lys Asn Trp Leu Ser Trp Tyr Gln Lys Lys Pro 50
55 60 Gly Gln Pro Pro Lys Leu
Leu Ile Tyr Lys Ala Ser Thr Leu Glu Ser 65 70
75 80 Gly Val Ser Ser Arg Phe Lys Gly Ser Gly Ser
Gly Thr Gln Phe Thr 85 90
95 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110 Gln Gly
Glu Phe Ser Cys Ser Ser Ala Asp Cys Thr Ala Phe Gly Gly 115
120 125 Gly Thr Glu Val Val Val Lys
Gly Asp Pro Val Ala Pro Thr Val Leu 130 135
140 Ile Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly
Thr Val Thr Ile 145 150 155
160 Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu
165 170 175 Val Asp Gly
Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro 180
185 190 Gln Asn Ser Ala Asp Cys Thr Tyr
Asn Leu Ser Ser Thr Leu Thr Leu 195 200
205 Thr Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys
Lys Val Thr 210 215 220
Gln Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225
230 235 38457PRTOryctolagus
cuniculus 38Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys
Gly 1 5 10 15 Val
Gln Cys Gln Ser Val Glu Glu Ser Gly Gly His Leu Val Thr Pro
20 25 30 Gly Thr Pro Leu Thr
Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35
40 45 Thr Tyr Ala Met Gly Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu 50 55
60 Trp Ile Gly Val Ile Thr Lys Ser Gly Asn Thr Tyr Tyr
Ala Ser Trp 65 70 75
80 Ala Lys Gly Arg Phe Ala Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
85 90 95 Lys Ile Thr Ser
Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Gly 100
105 110 Arg Tyr Gly Ile Gly Val Ser Tyr Tyr
Asp Ile Trp Gly Pro Gly Thr 115 120
125 Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val
Phe Pro 130 135 140
Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly 145
150 155 160 Cys Leu Val Lys Gly
Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn 165
170 175 Ser Gly Thr Leu Thr Asn Gly Val Arg Thr
Phe Pro Ser Val Arg Gln 180 185
190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser
Ser 195 200 205 Ser
Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys 210
215 220 Val Asp Lys Thr Val Ala
Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro 225 230
235 240 Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Ile Phe Pro Pro Lys 245 250
255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
260 265 270 Val Val
Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr 275
280 285 Ile Asn Asn Glu Gln Val Arg
Thr Ala Arg Pro Pro Leu Arg Glu Gln 290 295
300 Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu
Pro Ile Ala His 305 310 315
320 Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys
325 330 335 Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln 340
345 350 Pro Leu Glu Pro Lys Val Tyr Thr
Met Gly Pro Pro Arg Glu Glu Leu 355 360
365 Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly
Phe Tyr Pro 370 375 380
Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn 385
390 395 400 Tyr Lys Thr Thr
Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu 405
410 415 Tyr Ser Lys Leu Ser Val Pro Thr Ser
Glu Trp Gln Arg Gly Asp Val 420 425
430 Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 435 440 445
Lys Ser Ile Ser Arg Ser Pro Gly Lys 450 455
39134PRTOryctolagus cuniculus 39Met Asp Thr Arg Ala Pro Thr Gln Leu Leu
Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser
Pro 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser 35
40 45 Gln Ser Leu Tyr Asn Lys
Asn Trp Leu Ser Trp Tyr Gln Lys Lys Pro 50 55
60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Lys Ala
Ser Thr Leu Glu Ser 65 70 75
80 Gly Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95 Leu Thr
Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100
105 110 Gln Gly Glu Phe Ser Cys Ser
Ser Ala Asp Cys Thr Ala Phe Gly Gly 115 120
125 Gly Thr Glu Val Val Val 130
40136PRTOryctolagus cuniculus 40Met Glu Thr Gly Leu Arg Trp Leu Leu Leu
Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly His Leu Val Thr
Pro 20 25 30 Gly
Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35
40 45 Thr Tyr Ala Met Gly Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Trp Ile Gly Val Ile Thr Lys Ser Gly Asn Thr
Tyr Tyr Ala Ser Trp 65 70 75
80 Ala Lys Gly Arg Phe Ala Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
85 90 95 Lys Ile
Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Gly 100
105 110 Arg Tyr Gly Ile Gly Val Ser
Tyr Tyr Asp Ile Trp Gly Pro Gly Thr 115 120
125 Leu Val Thr Val Ser Ser Gly Gln 130
135 415PRTOryctolagus cuniculus 41Thr Tyr Ala Met Gly 1
5 4216PRTOryctolagus cuniculus 42Val Ile Thr Lys Ser Gly Asn
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5
10 15 4310PRTOryctolagus cuniculus 43Tyr Gly Ile
Gly Val Ser Tyr Tyr Asp Ile 1 5 10
4413PRTOryctolagus cuniculus 44Gln Ser Ser Gln Ser Leu Tyr Asn Lys Asn
Trp Leu Ser 1 5 10
457PRTOryctolagus cuniculus 45Lys Ala Ser Thr Leu Glu Ser 1
5 4613PRTOryctolagus cuniculus 46Gln Gly Glu Phe Ser Cys Ser
Ser Ala Asp Cys Thr Ala 1 5 10
477PRTHomo Sapien 47Asn Gly Arg Tyr Ser Ile Ser 1 5
485PRTHomo Sapien 48Glu Gly His Val Val 1 5
499PRTHomo Sapien 49Thr Ser Asn Thr Ser Asn Tyr Asp Thr 1 5
5011PRTHomo Sapien 50Asn Arg Asp Gly Thr Arg Tyr Val
Gln Lys Gly 1 5 10 5118PRTHomo
Sapien 51Ala Asn Asn Thr Phe Val Tyr Ile Leu Thr Ser Asn Thr Ser Asn Tyr
1 5 10 15 Asp Thr
5211PRTHomo Sapien 52Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr 1
5 10 5311PRTHomo Sapien 53Asn Asp Cys Pro
Gly Pro Gly Gln Asp Thr Asp 1 5 10
547PRTHomo Sapien 54Tyr Trp Ser Glu Asn Leu Phe 1 5
559PRTHomo Sapien 55His Leu Ser Cys Gln Glu Lys Gln Asn 1
5 5615PRTHomo Sapien 56Cys Thr Cys His Ala Gly Phe Phe
Leu Arg Glu Asn Glu Cys Val 1 5 10
15 577PRTHomo Sapien 57Cys Glu His Ala Gly Lys Cys 1
5 589PRTHomo Sapien 58Met Pro Gly Tyr Glu Gly Val His Cys 1
5 597PRTHomo Sapien 59Cys Leu His Asn Gly
Arg Cys 1 5 605PRTHomo Sapien 60Lys Ile Asn Glu
Phe 1 5 6125PRTHomo Sapien 61Glu Cys Ala Ser Ser Pro Cys
Leu His Asn Gly Arg Cys Leu Asp Lys 1 5
10 15 Ile Asn Glu Phe Gln Cys Glu Cys Pro
20 25 629PRTHomo Sapien 62Leu His His Asp Gly Gln Phe
Cys His 1 5 6311PRTHomo Sapien 63Ala Arg
Asp Cys Thr Val Asn Gly Asp Glu Pro 1 5
10 647PRTHomo Sapien 64Arg Leu Cys Asp Glu Gly His 1 5
657PRTHomo Sapien 65Asn Ser Thr Val Cys Glu His 1
5 6613PRTHomo Sapien 66Glu Glu Pro Ser Arg Ser Asn Leu Gly Trp
Leu Cys Leu 1 5 10
6715PRTHomo Sapien 67Ser Asn Lys Leu Thr Gln Leu Phe Thr Phe Glu Asp His
Phe Leu 1 5 10 15
6811PRTHomo Sapien 68Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 1
5 10 6913PRTHomo Sapien 69Ile Arg Gly Asn
Met Tyr Tyr Glu Asn Ser Tyr Ala Leu 1 5
10 7011PRTHomo Sapien 70Val Leu Ser Asn Tyr Asp Ala Asn Lys
Thr Gly 1 5 10 7113PRTHomo Sapien
71Ile Val Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp 1 5
10 7215PRTHomo Sapien 72Phe Leu Ser Asn Met
Ser Met Asp Phe Gln Asn His Leu Gly Ser 1 5
10 15 736PRTHomo Sapien 73Trp Gly Ala Gly Glu Glu 1
5 7418PRTHomo Sapien 74Pro Pro Leu Met Leu Tyr Asn Pro
Thr Thr Tyr Gln Met Asp Val Asn 1 5 10
15 Pro Glu 7510PRTHomo Sapien 75Pro Glu Gly Lys Tyr
Ser Phe Gly Ala Thr 1 5 10 7610PRTHomo
Sapien 76Arg Asn Tyr Val Val Thr Asp His Gly Ser 1 5
10 7714PRTHomo Sapien 77Gly Ala Asp Ser Tyr Glu Met Glu Glu
Asp Gly Val Arg Lys 1 5 10
7815PRTHomo Sapien 78Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
Ser Ile Asn 1 5 10 15
799PRTHomo Sapien 79Ala Thr Asn Ile Lys His Phe Lys Asn 1 5
8016PRTHomo Sapien 80Leu Pro Val Ala Phe Arg Gly Asp
Ser Phe Thr His Thr Pro Pro Leu 1 5 10
15 8110PRTHomo Sapien 81Lys Ile Ile Ser Asn Arg Gly
Glu Asn Ser 1 5 10 8215PRTHomo Sapien
82Tyr Thr Glu Ile His Pro Glu Met Arg His Val Asp Cys Gln Ser 1
5 10 15 839PRTHomo Sapien 83Gly
Glu Phe Ala Thr Ser Lys Ile Asn 1 5
847PRTHomo Sapien 84Trp Arg Lys Asp Cys Ser Asn 1 5
8511PRTHomo Sapien 85Gly Ser Arg Ser Lys Ile Phe Asp Lys Asp Ser 1
5 10 8611PRTHomo Sapien 86Ile His Gly Gly
Arg Glu Asp Ser Arg Asp Leu 1 5 10
871374DNAOryctolagus cuniculus 87atggagactg ggctgcgctg gcttctcctg
gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg tcacctggtc
acgcctggga cacccctgac actcacctgc 120acagtctctg gaatcgacct cagtacctat
gcaatgggct gggtccgcca ggctccaggg 180aaggggctgg agtggatcgg agtcattact
aaaagtggta acacatacta cgcgagctgg 240gcgaaaggcc gattcgccat ctccaaaacc
tcgaccacgg tggatctaaa gatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtggcagat atggtattgg tgtttcttac 360tatgacatct ggggcccagg cactctggtc
accgtctcct cagggcaacc taaggctcca 420tcagtcttcc cactggcccc ctgctgcggg
gacacaccca gctccacggt gaccctgggc 480tgcctggtca aagggtacct cccggagcca
gtgaccgtga cctggaactc gggcaccctc 540accaatgggg tacgcacctt cccgtccgtc
cggcagtcct caggcctcta ctcgctgagc 600agcgtggtga gcgtgacctc aagcagccag
cccgtcacct gcaacgtggc ccacccagcc 660accaacacca aagtggacaa gaccgttgcg
ccctcgacat gcagcaagcc cacgtgccca 720ccccctgaac tcctgggggg accgtctgtc
ttcatcttcc ccccaaaacc caaggacacc 780ctcatgatct cacgcacccc cgaggtcaca
tgcgtggtgg tggacgtgag ccaggatgac 840cccgaggtgc agttcacatg gtacataaac
aacgagcagg tgcgcaccgc ccggccgccg 900ctacgggagc agcagttcaa cagcacgatc
cgcgtggtca gcaccctccc catcgcgcac 960caggactggc tgaggggcaa ggagttcaag
tgcaaagtcc acaacaaggc actcccggcc 1020cccatcgaga aaaccatctc caaagccaga
gggcagcccc tggagccgaa ggtctacacc 1080atgggccctc cccgggagga gctgagcagc
aggtcggtca gcctgacctg catgatcaac 1140ggcttctacc cttccgacat ctcggtggag
tgggagaaga acgggaaggc agaggacaac 1200tacaagacca cgccggccgt gctggacagc
gacggctcct acttcctcta cagcaagctc 1260tcagtgccca cgagtgagtg gcagcggggc
gacgtcttca cctgctccgt gatgcacgag 1320gccttgcaca accactacac gcagaagtcc
atctcccgct ctccgggtaa atga 137488720DNAOryctolagus cuniculus
88atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60acatttgccc aagtgctgac ccagactcca tcccctgtgt ctgcagctgt gggaggcaca
120gtcaccatca attgccagtc cagtcagagt ctttataata agaactggtt atcctggtat
180cagaagaaac cagggcagcc tcctaagctc ctgatctaca aggcatccac tctggaatct
240ggggtctcat cgcggttcaa gggcagtgga tctgggacac agttcactct caccatcagc
300ggcgtgcagt gtgacgatgc tgccacttac tactgtcaag gcgaatttag ttgtagtagt
360gctgattgta cggctttcgg cggagggacc gaggtggtgg tcaaaggtga tccagttgca
420cctactgtcc tcatcttccc accagctgct gatcaggtgg caactggaac agtcaccatc
480gtgtgtgtgg cgaataaata ctttcccgat gtcaccgtca cctgggaggt ggatggcacc
540acccaaacaa ctggcatcga gaacagtaaa acaccgcaga attctgcaga ttgtacctac
600aacctcagca gcactctgac actgaccagc acacagtaca acagccacaa agagtacacc
660tgcaaggtga cccagggcac gacctcagtc gtccagagct tcaatagggg tgactgttag
72089456PRTOryctolagus cuniculus 89Met Glu Thr Gly Leu Arg Trp Leu Leu
Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val
Thr Pro 20 25 30
Gly Thr Pro Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Ser Leu Ser
35 40 45 Asp Ser Arg Val
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50
55 60 Trp Ile Gly Ile Val Gly Ile Gly
Trp Asn Ile Tyr His Ala Asn Trp 65 70
75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser
Thr Thr Val Asp 85 90
95 Leu Lys Ile Thr Ser Pro Thr Val Glu Asp Thr Ala Thr Tyr Phe Cys
100 105 110 Ala Arg Gly
Leu Gly Gly Gly Thr Val Ile Trp Gly Pro Gly Thr Leu 115
120 125 Val Thr Val Ser Leu Gly Gln Pro
Lys Ala Pro Ser Val Phe Pro Leu 130 135
140 Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr
Leu Gly Cys 145 150 155
160 Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser
165 170 175 Gly Thr Leu Thr
Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser 180
185 190 Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Ser Val Thr Ser Ser Ser 195 200
205 Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr
Lys Val 210 215 220
Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro 225
230 235 240 Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro 245
250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val 260 265
270 Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr
Ile 275 280 285 Asn
Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln 290
295 300 Phe Asn Ser Thr Ile Arg
Val Val Ser Thr Leu Pro Ile Ala His Gln 305 310
315 320 Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys
Val His Asn Lys Ala 325 330
335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro
340 345 350 Leu Glu
Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser 355
360 365 Ser Arg Ser Val Ser Leu Thr
Cys Met Ile Asn Gly Phe Tyr Pro Ser 370 375
380 Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala
Glu Asp Asn Tyr 385 390 395
400 Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr
405 410 415 Ser Lys Leu
Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe 420
425 430 Thr Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys 435 440
445 Ser Ile Ser Arg Ser Pro Gly Lys 450
455 90133PRTOryctolagus cuniculus 90Met Glu Thr Gly Leu Arg Trp
Leu Leu Leu Val Ala Val Leu Lys Gly 1 5
10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25
30 Gly Thr Pro Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Ser Leu
Ser 35 40 45 Asp
Ser Arg Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50
55 60 Trp Ile Gly Ile Val Gly
Ile Gly Trp Asn Ile Tyr His Ala Asn Trp 65 70
75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser
Ser Thr Thr Val Asp 85 90
95 Leu Lys Ile Thr Ser Pro Thr Val Glu Asp Thr Ala Thr Tyr Phe Cys
100 105 110 Ala Arg
Gly Leu Gly Gly Gly Thr Val Ile Trp Gly Pro Gly Thr Leu 115
120 125 Val Thr Val Ser Leu 130
91239PRTOryctolagus cuniculus 91Met Asp Thr Arg Ala Pro Thr
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr
Gln Thr Pro Ala Ser 20 25
30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser
Ser 35 40 45 Glu
Ser Val Tyr Lys Asn Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro 50
55 60 Gly Gln Pro Pro Lys Leu
Leu Ile Tyr Glu Ala Ser Lys Leu Ala Ser 65 70
75 80 Gly Val Ser Thr Arg Phe Lys Gly Ser Gly Ser
Gly Thr Gln Phe Thr 85 90
95 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110 Leu Gly
Glu Phe Ser Cys Tyr Ser Gly Asp Cys Gly Thr Phe Gly Gly 115
120 125 Gly Thr Ala Val Val Val Lys
Gly Asp Pro Val Ala Pro Thr Val Leu 130 135
140 Ile Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly
Thr Val Thr Ile 145 150 155
160 Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu
165 170 175 Val Asp Gly
Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro 180
185 190 Gln Asn Ser Ala Asp Cys Thr Tyr
Asn Leu Ser Ser Thr Leu Thr Leu 195 200
205 Thr Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys
Lys Val Thr 210 215 220
Gln Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225
230 235 92135PRTOryctolagus
cuniculus 92Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu
Trp 1 5 10 15 Leu
Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ala Ser
20 25 30 Val Ser Ala Ala Val
Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser 35
40 45 Glu Ser Val Tyr Lys Asn Asn Tyr Leu
Ser Trp Phe Gln Gln Lys Pro 50 55
60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys
Leu Ala Ser 65 70 75
80 Gly Val Ser Thr Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95 Leu Thr Ile Ser
Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100
105 110 Leu Gly Glu Phe Ser Cys Tyr Ser Gly
Asp Cys Gly Thr Phe Gly Gly 115 120
125 Gly Thr Ala Val Val Val Lys 130 135
935PRTOryctolagus cuniculus 93Asp Ser Arg Val Ser 1 5
9416PRTOryctolagus cuniculus 94Ile Val Gly Ile Gly Trp Asn Ile Tyr His
Ala Asn Trp Ala Lys Gly 1 5 10
15 958PRTOryctolagus cuniculus 95Gly Leu Gly Gly Gly Thr Val
Ile 1 5 9612PRTOryctolagus cuniculus 96Gln
Ser Ser Glu Ser Val Lys Asn Asn Tyr Leu Ser 1 5
10 977PRTOryctolagus cuniculus 97Glu Ala Ser Lys Leu Ala
Ser 1 5 9813PRTOryctolagus cuniculus 98Leu Gly
Glu Phe Ser Cys Tyr Ser Gly Asp Cys Gly Thr 1 5
10 991371DNAOryctolagus cuniculus 99atggagactg
ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg
agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120aaagcctctg
gattctccct cagtgactct agagtgagct gggtccgcca ggctccaggg 180aaggggctgg
aatggatcgg aatcgttggc attggttgga atatatacca cgcgaactgg 240gcgaaaggcc
gattcaccat ctccaaaacg tcgtcgacca cggtggattt gaaaatcacc 300agtccgacag
tcgaggacac ggccacctat ttctgtgcca gaggtctggg tggtggtact 360gtcatctggg
gcccaggcac cctggtcacc gtctccttag ggcaacctaa ggctccatca 420gtcttcccac
tggccccctg ctgcggggac acacccagct ccacggtgac cctgggctgc 480ctggtcaaag
ggtacctccc ggagccagtg accgtgacct ggaactcggg caccctcacc 540aatggggtac
gcaccttccc gtccgtccgg cagtcctcag gcctctactc gctgagcagc 600gtggtgagcg
tgacctcaag cagccagccc gtcacctgca acgtggccca cccagccacc 660aacaccaaag
tggacaagac cgttgcgccc tcgacatgca gcaagcccac gtgcccaccc 720cctgaactcc
tggggggacc gtctgtcttc atcttccccc caaaacccaa ggacaccctc 780atgatctcac
gcacccccga ggtcacatgc gtggtggtgg acgtgagcca ggatgacccc 840gaggtgcagt
tcacatggta cataaacaac gagcaggtgc gcaccgcccg gccgccgcta 900cgggagcagc
agttcaacag cacgatccgc gtggtcagca ccctccccat cgcgcaccag 960gactggctga
ggggcaagga gttcaagtgc aaagtccaca acaaggcact cccggccccc 1020atcgagaaaa
ccatctccaa agccagaggg cagcccctgg agccgaaggt ctacaccatg 1080ggccctcccc
gggaggagct gagcagcagg tcggtcagcc tgacctgcat gatcaacggc 1140ttctaccctt
ccgacatctc ggtggagtgg gagaagaacg ggaaggcaga ggacaactac 1200aagaccacgc
cggccgtgct ggacagcgac ggctcctact tcctctacag caagctctca 1260gtgcccacga
gtgagtggca gcggggcgac gtcttcacct gctccgtgat gcacgaggcc 1320ttgcacaacc
actacacgca gaagtccatc tcccgctctc cgggtaaatg a
1371100399DNAOryctolagus cuniculus 100atggagactg ggctgcgctg gcttctcctg
gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc
acgcctggga cacccctgac actcacctgc 120aaagcctctg gattctccct cagtgactct
agagtgagct gggtccgcca ggctccaggg 180aaggggctgg aatggatcgg aatcgttggc
attggttgga atatatacca cgcgaactgg 240gcgaaaggcc gattcaccat ctccaaaacg
tcgtcgacca cggtggattt gaaaatcacc 300agtccgacag tcgaggacac ggccacctat
ttctgtgcca gaggtctggg tggtggtact 360gtcatctggg gcccaggcac cctggtcacc
gtctcctta 399101720DNAOryctolagus cuniculus
101atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60acatttgccc aagtgctgac ccagactcca gcctccgtgt ctgcagctgt gggaggcaca
120gtcaccatca gttgccagtc cagtgagagt gtttataaga acaactactt atcctggttt
180cagcagaaac caggacagcc tcccaagctc ctgatctacg aagcatccaa actggcatct
240ggggtctcaa cgcggttcaa aggcagtgga tctgggacac agttcactct caccatcagc
300ggcgtgcagt gtgacgatgc tgccacatac tactgtctag gcgaatttag ttgttatagt
360ggtgattgtg gtactttcgg cggagggacc gcggtggtgg tcaaaggtga tccagttgca
420cctactgtcc tcatcttccc accagctgct gatcaggtgg caactggaac agtcaccatc
480gtgtgtgtgg cgaataaata ctttcccgat gtcaccgtca cctgggaggt ggatggcacc
540acccaaacaa ctggcatcga gaacagtaaa acaccgcaga attctgcaga ttgtacctac
600aacctcagca gcactctgac actgaccagc acacagtaca acagccacaa agagtacacc
660tgcaaggtga cccagggcac gacctcagtc gtccagagct tcaatagggg tgactgttag
720102405DNAOryctolagus cuniculus 102atggacacga gggcccccac tcagctgctg
gggctcctgc tgctctggct cccaggtgcc 60acatttgccc aagtgctgac ccagactcca
gcctccgtgt ctgcagctgt gggaggcaca 120gtcaccatca gttgccagtc cagtgagagt
gtttataaga acaactactt atcctggttt 180cagcagaaac caggacagcc tcccaagctc
ctgatctacg aagcatccaa actggcatct 240ggggtctcaa cgcggttcaa aggcagtgga
tctgggacac agttcactct caccatcagc 300ggcgtgcagt gtgacgatgc tgccacatac
tactgtctag gcgaatttag ttgttatagt 360ggtgattgtg gtactttcgg cggagggacc
gcggtggtgg tcaaa 405103458PRTOryctolagus cuniculus
103Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1
5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20
25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe Ser Leu Ser 35 40
45 Arg Asn Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu 50 55 60
Tyr Ile Gly Ile Ile Gly Ser Ser Gly Val Thr Tyr Tyr Ala Ser Trp 65
70 75 80 Ala Lys Gly Arg Phe
Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu 85
90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Ala 100 105
110 Arg Asn Leu Tyr Thr Gly Gly Ser Asn Asp Asn Leu Trp Gly Pro
Gly 115 120 125 Thr
Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe 130
135 140 Pro Leu Ala Pro Cys Cys
Gly Asp Thr Pro Ser Ser Thr Val Thr Leu 145 150
155 160 Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro
Val Thr Val Thr Trp 165 170
175 Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg
180 185 190 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser 195
200 205 Ser Ser Gln Pro Val Thr Cys
Asn Val Ala His Pro Ala Thr Asn Thr 210 215
220 Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser
Lys Pro Thr Cys 225 230 235
240 Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
245 250 255 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 260
265 270 Val Val Val Asp Val Ser Gln Asp
Asp Pro Glu Val Gln Phe Thr Trp 275 280
285 Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro
Leu Arg Glu 290 295 300
Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala 305
310 315 320 His Gln Asp Trp
Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn 325
330 335 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Arg Gly 340 345
350 Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg
Glu Glu 355 360 365
Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr 370
375 380 Pro Ser Asp Ile Ser
Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp 385 390
395 400 Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp
Ser Asp Gly Ser Tyr Phe 405 410
415 Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly
Asp 420 425 430 Val
Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435
440 445 Gln Lys Ser Ile Ser Arg
Ser Pro Gly Lys 450 455
104135PRTOryctolagus cuniculus 104Met Glu Thr Gly Leu Arg Trp Leu Leu Leu
Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr
Pro 20 25 30 Gly
Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35
40 45 Arg Asn Ala Ile Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Tyr Ile Gly Ile Ile Gly Ser Ser Gly Val Thr
Tyr Tyr Ala Ser Trp 65 70 75
80 Ala Lys Gly Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu
85 90 95 Lys Ile
Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100
105 110 Arg Asn Leu Tyr Thr Gly Gly
Ser Asn Asp Asn Leu Trp Gly Pro Gly 115 120
125 Thr Leu Val Thr Val Ser Ser 130
135 105237PRTOryctolagus cuniculus 105Met Asp Thr Arg Val Pro Thr Gln
Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr
Pro Ser Pro 20 25 30
Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser
35 40 45 Lys Ser Val Tyr
Asn Asn Val Gln Leu Ser Trp Phe Gln Gln Lys Pro 50
55 60 Gly Gln Pro Pro Lys Arg Leu Ile
Tyr Tyr Ala Ser Thr Leu Ala Ser 65 70
75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly
Thr Gln Phe Thr 85 90
95 Leu Thr Ile Ser Asp Val Gln Cys Asp Asp Val Ala Thr Tyr Tyr Cys
100 105 110 Ala Gly Gly
Tyr Ser Ser Ser Ser Asp Asn Ala Phe Gly Gly Gly Thr 115
120 125 Glu Val Val Val Lys Gly Asp Pro
Val Ala Pro Thr Val Leu Ile Phe 130 135
140 Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr
Ile Val Cys 145 150 155
160 Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp
165 170 175 Gly Thr Thr Gln
Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn 180
185 190 Ser Ala Asp Cys Thr Tyr Asn Leu Ser
Ser Thr Leu Thr Leu Thr Ser 195 200
205 Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr
Gln Gly 210 215 220
Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225
230 235 106133PRTOryctolagus cuniculus 106Met Asp
Thr Arg Val Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Pro Gly Ala Thr Phe Ala
Gln Val Leu Thr Gln Thr Pro Ser Pro 20 25
30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn
Cys Gln Ala Ser 35 40 45
Lys Ser Val Tyr Asn Asn Val Gln Leu Ser Trp Phe Gln Gln Lys Pro
50 55 60 Gly Gln Pro
Pro Lys Arg Leu Ile Tyr Tyr Ala Ser Thr Leu Ala Ser 65
70 75 80 Gly Val Pro Ser Arg Phe Lys
Gly Ser Gly Ser Gly Thr Gln Phe Thr 85
90 95 Leu Thr Ile Ser Asp Val Gln Cys Asp Asp Val
Ala Thr Tyr Tyr Cys 100 105
110 Ala Gly Gly Tyr Ser Ser Ser Ser Asp Asn Ala Phe Gly Gly Gly
Thr 115 120 125 Glu
Val Val Val Lys 130 1075PRTOryctolagus cuniculus 107Arg
Asn Ala Ile Asn 1 5 10816PRTOryctolagus cuniculus 108Ile
Ile Gly Ser Ser Gly Val Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1
5 10 15 10911PRTOryctolagus
cuniculus 109Asn Leu Tyr Thr Gly Gly Ser Asn Asp Asn Leu 1
5 10 11013PRTOryctolagus cuniculus 110Gln Ala Ser
Lys Ser Val Tyr Asn Asn Val Gln Leu Ser 1 5
10 1117PRTOryctolagus cuniculus 111Tyr Ala Ser Thr Leu Ala
Ser 1 5 11211PRTOryctolagus cuniculus 112Ala Gly
Gly Tyr Ser Ser Ser Ser Asp Asn Ala 1 5
10 1131377DNAOryctolagus cuniculus 113atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgttggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120accgtctctg gattctccct
cagtcgcaat gcaataaact gggtccgcca ggctccaggg 180aaggggctgg aatacatcgg
aatcattggt agtagtggtg tcacatacta cgcgagctgg 240gcaaaaggcc gattcaccat
ctccagaacc tcgaccacgg tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc
cacctatttt tgtgccagaa atctttatac tggtggtagt 360aatgataact tgtggggccc
aggcaccctg gtcaccgtct cctcagggca acctaaggct 420ccatcagtct tcccactggc
cccctgctgc ggggacacac ccagctccac ggtgaccctg 480ggctgcctgg tcaaagggta
cctcccggag ccagtgaccg tgacctggaa ctcgggcacc 540ctcaccaatg gggtacgcac
cttcccgtcc gtccggcagt cctcaggcct ctactcgctg 600agcagcgtgg tgagcgtgac
ctcaagcagc cagcccgtca cctgcaacgt ggcccaccca 660gccaccaaca ccaaagtgga
caagaccgtt gcgccctcga catgcagcaa gcccacgtgc 720ccaccccctg aactcctggg
gggaccgtct gtcttcatct tccccccaaa acccaaggac 780accctcatga tctcacgcac
ccccgaggtc acatgcgtgg tggtggacgt gagccaggat 840gaccccgagg tgcagttcac
atggtacata aacaacgagc aggtgcgcac cgcccggccg 900ccgctacggg agcagcagtt
caacagcacg atccgcgtgg tcagcaccct ccccatcgcg 960caccaggact ggctgagggg
caaggagttc aagtgcaaag tccacaacaa ggcactcccg 1020gcccccatcg agaaaaccat
ctccaaagcc agagggcagc ccctggagcc gaaggtctac 1080accatgggcc ctccccggga
ggagctgagc agcaggtcgg tcagcctgac ctgcatgatc 1140aacggcttct acccttccga
catctcggtg gagtgggaga agaacgggaa ggcagaggac 1200aactacaaga ccacgccggc
cgtgctggac agcgacggct cctacttcct ctacagcaag 1260ctctcagtgc ccacgagtga
gtggcagcgg ggcgacgtct tcacctgctc cgtgatgcac 1320gaggccttgc acaaccacta
cacgcagaag tccatctccc gctctccggg taaatga 1377114405DNAOryctolagus
cuniculus 114atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgttggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120accgtctctg gattctccct cagtcgcaat gcaataaact gggtccgcca
ggctccaggg 180aaggggctgg aatacatcgg aatcattggt agtagtggtg tcacatacta
cgcgagctgg 240gcaaaaggcc gattcaccat ctccagaacc tcgaccacgg tggatctgaa
aatcaccagt 300ccgacaaccg aggacacggc cacctatttt tgtgccagaa atctttatac
tggtggtagt 360aatgataact tgtggggccc aggcaccctg gtcaccgtct cctca
4051151371DNAOryctolagus cuniculus 115atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120aaagcctctg gattctccct
cagtgactct agagtgagct gggtccgcca ggctccaggg 180aaggggctgg aatggatcgg
aatcgttggc attggttgga atatatacca cgcgaactgg 240gcgaaaggcc gattcaccat
ctccaaaacg tcgtcgacca cggtggattt gaaaatcacc 300agtccgacag tcgaggacac
ggccacctat ttctgtgcca gaggtctggg tggtggtact 360gtcatctggg gcccaggcac
cctggtcacc gtctccttag ggcaacctaa ggctccatca 420gtcttcccac tggccccctg
ctgcggggac acacccagct ccacggtgac cctgggctgc 480ctggtcaaag ggtacctccc
ggagccagtg accgtgacct ggaactcggg caccctcacc 540aatggggtac gcaccttccc
gtccgtccgg cagtcctcag gcctctactc gctgagcagc 600gtggtgagcg tgacctcaag
cagccagccc gtcacctgca acgtggccca cccagccacc 660aacaccaaag tggacaagac
cgttgcgccc tcgacatgca gcaagcccac gtgcccaccc 720cctgaactcc tggggggacc
gtctgtcttc atcttccccc caaaacccaa ggacaccctc 780atgatctcac gcacccccga
ggtcacatgc gtggtggtgg acgtgagcca ggatgacccc 840gaggtgcagt tcacatggta
cataaacaac gagcaggtgc gcaccgcccg gccgccgcta 900cgggagcagc agttcaacag
cacgatccgc gtggtcagca ccctccccat cgcgcaccag 960gactggctga ggggcaagga
gttcaagtgc aaagtccaca acaaggcact cccggccccc 1020atcgagaaaa ccatctccaa
agccagaggg cagcccctgg agccgaaggt ctacaccatg 1080ggccctcccc gggaggagct
gagcagcagg tcggtcagcc tgacctgcat gatcaacggc 1140ttctaccctt ccgacatctc
ggtggagtgg gagaagaacg ggaaggcaga ggacaactac 1200aagaccacgc cggccgtgct
ggacagcgac ggctcctact tcctctacag caagctctca 1260gtgcccacga gtgagtggca
gcggggcgac gtcttcacct gctccgtgat gcacgaggcc 1320ttgcacaacc actacacgca
gaagtccatc tcccgctctc cgggtaaatg a 1371116399DNAOryctolagus
cuniculus 116atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120aaagcctctg gattctccct cagtgactct agagtgagct gggtccgcca
ggctccaggg 180aaggggctgg aatggatcgg aatcgttggc attggttgga atatatacca
cgcgaactgg 240gcgaaaggcc gattcaccat ctccaaaacg tcgtcgacca cggtggattt
gaaaatcacc 300agtccgacag tcgaggacac ggccacctat ttctgtgcca gaggtctggg
tggtggtact 360gtcatctggg gcccaggcac cctggtcacc gtctcctta
3991175PRTArtificial Sequencelinker 117Ser Gly Gly Gly Gly 1
5 11813PRTArtificial SequencePADRE Epitope 118Ala Lys Phe
Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 1 5
10 1191418DNAArtificial SequenceSynthetic Construct
119aagcttgtac ccttcaccat ggagactggg ctgcgctggc ttctcctggt cgctgtgctc
60aaaggtgtcc agtgtcagtc ggtggaggag tccgggggtc acctggtcac gcctgggaca
120cccctgacac tcacctgcac agtctctgga atcgacctca gtacctatgc aatgggctgg
180gtccgccagg ctccagggaa ggggctggag tggatcggag tcattactaa aagtggtaac
240acatactacg cgagctgggc gaaaggccga ttcgccatct ccaaaacctc gaccacggtg
300gatctaaaga tcaccagtcc gacaaccgag gacacggcca cctatttctg tggcagatat
360ggtattggtg tttcttacta tgacatctgg ggcccaggca ctctggtcac cgtctcctca
420gggcaaccta aggctccatc agtcttccca ctggccccct gctgcgggga cacacccagc
480tccacggtga ccctgggctg cctggtcaaa gggtacctcc cggagccagt gaccgtgacc
540tggaactcgg gcaccctcac caatggggta cgcaccttcc cgtccgtccg gcagtcctca
600ggcctctact cgctgagcag cgtggtgagc gtgacctcaa gcagccagcc cgtcacctgc
660aacgtggccc acccagccac caacaccaaa gtggacaaga ccgttgcgcc ctcgacatgc
720agcaagccca cgtgcccacc ccctgaactc ctggggggac cgtctgtctt catcttcccc
780ccaaaaccca aggacaccct catgatctca cgcacccccg aggtcacatg cgtggtggtg
840gacgtgagcc aggatgaccc cgaggtgcag ttcacatggt acataaacaa cgagcaggtg
900cgcaccgccc ggccgccgct acgggagcag cagttcaaca gcacgatccg cgtggtcagc
960accctcccca tcgcgcacca ggactggctg aggggcaagg agttcaagtg caaagtccac
1020aacaaggcac tcccggcccc catcgagaaa accatctcca aagccagagg gcagcccctg
1080gagccgaagg tctacaccat gggccctccc cgggaggagc tgagcagcag gtcggtcagc
1140ctgacctgca tgatcaacgg cttctaccct tccgacatct cggtggagtg ggagaagaac
1200gggaaggcag aggacaacta caagaccacg ccggccgtgc tggacagcga cggctcctac
1260ttcctctaca gcaagctctc agtgcccacg agtgagtggc agcggggcga cgtcttcacc
1320tgctccgtga tgcacgaggc cttgcacaac cactacacgc agaagtccat ctcccgctct
1380ccgggtaaat gagcgctgtg ccggcgagct gcggccgc
1418120753DNAArtificial SequenceSynthetic Construct 120aagcttgtac
ccttcaccat ggacacgagg gcccccactc agctgctggg gctcctgctg 60ctctggctcc
caggtgccac atttgcccaa gtgctgaccc agactccatc ccctgtgtct 120gcagctgtgg
gaggcacagt caccatcaat tgccagtcca gtcagagtct ttataataag 180aactggttat
cctggtatca gaagaaacca gggcagcctc ctaagctcct gatctacaag 240gcatccactc
tggaatctgg ggtctcatcg cggttcaagg gcagtggatc tgggacacag 300ttcactctca
ccatcagcgg cgtgcagtgt gacgatgctg ccacttacta ctgtcaaggc 360gaatttagtt
gtagtagtgc tgattgtacg gctttcggcg gagggaccga ggtggtggtc 420aaaggtgatc
cagttgcacc tactgtcctc atcttcccac cagctgctga tcaggtggca 480actggaacag
tcaccatcgt gtgtgtggcg aataaatact ttcccgatgt caccgtcacc 540tgggaggtgg
atggcaccac ccaaacaact ggcatcgaga acagtaaaac accgcagaat 600tctgcagatt
gtacctacaa cctcagcagc actctgacac tgaccagcac acagtacaac 660agccacaaag
agtacacctg caaggtgacc cagggcacga cctcagtcgt ccagagcttc 720aataggggtg
actgttagag tgagagcggc cgc
7531211415DNAArtificial SequenceSynthetic Construct 121aagcttgtac
ccttcaccat ggagactggg ctgcgctggc ttctcctggt cgctgtgctc 60aaaggtgtcc
agtgtcagtc ggtggaggag tccgggggtc gcctggtcac gcctgggaca 120cccctgacac
tcacctgcaa agcctctgga ttctccctca gtgactctag agtgagctgg 180gtccgccagg
ctccagggaa ggggctggaa tggatcggaa tcgttggcat tggttggaat 240atataccacg
cgaactgggc gaaaggccga ttcaccatct ccaaaacgtc gtcgaccacg 300gtggatttga
aaatcaccag tccgacagtc gaggacacgg ccacctattt ctgtgccaga 360ggtctgggtg
gtggtactgt catctggggc ccaggcaccc tggtcaccgt ctccttaggg 420caacctaagg
ctccatcagt cttcccactg gccccctgct gcggggacac acccagctcc 480acggtgaccc
tgggctgcct ggtcaaaggg tacctcccgg agccagtgac cgtgacctgg 540aactcgggca
ccctcaccaa tggggtacgc accttcccgt ccgtccggca gtcctcaggc 600ctctactcgc
tgagcagcgt ggtgagcgtg acctcaagca gccagcccgt cacctgcaac 660gtggcccacc
cagccaccaa caccaaagtg gacaagaccg ttgcgccctc gacatgcagc 720aagcccacgt
gcccaccccc tgaactcctg gggggaccgt ctgtcttcat cttcccccca 780aaacccaagg
acaccctcat gatctcacgc acccccgagg tcacatgcgt ggtggtggac 840gtgagccagg
atgaccccga ggtgcagttc acatggtaca taaacaacga gcaggtgcgc 900accgcccggc
cgccgctacg ggagcagcag ttcaacagca cgatccgcgt ggtcagcacc 960ctccccatcg
cgcaccagga ctggctgagg ggcaaggagt tcaagtgcaa agtccacaac 1020aaggcactcc
cggcccccat cgagaaaacc atctccaaag ccagagggca gcccctggag 1080ccgaaggtct
acaccatggg ccctccccgg gaggagctga gcagcaggtc ggtcagcctg 1140acctgcatga
tcaacggctt ctacccttcc gacatctcgg tggagtggga gaagaacggg 1200aaggcagagg
acaactacaa gaccacgccg gccgtgctgg acagcgacgg ctcctacttc 1260ctctacagca
agctctcagt gcccacgagt gagtggcagc ggggcgacgt cttcacctgc 1320tccgtgatgc
acgaggcctt gcacaaccac tacacgcaga agtccatctc ccgctctccg 1380ggtaaatgag
cgctgtgccg gcgagctgcg gccgc
1415122753DNAArtificial SequenceSynthetic Construct 122aagcttgtac
ccttcaccat ggacacgagg gcccccactc agctgctggg gctcctgctg 60ctctggctcc
caggtgccac atttgcccaa gtgctgaccc agactccagc ctccgtgtct 120gcagctgtgg
gaggcacagt caccatcagt tgccagtcca gtgagagtgt ttataagaac 180aactacttat
cctggtttca gcagaaacca ggacagcctc ccaagctcct gatctacgaa 240gcatccaaac
tggcatctgg ggtctcaacg cggttcaaag gcagtggatc tgggacacag 300ttcactctca
ccatcagcgg cgtgcagtgt gacgatgctg ccacatacta ctgtctaggc 360gaatttagtt
gttatagtgg tgattgtggt actttcggcg gagggaccgc ggtggtggtc 420aaaggtgatc
cagttgcacc tactgtcctc atcttcccac cagctgctga tcaggtggca 480actggaacag
tcaccatcgt gtgtgtggcg aataaatact ttcccgatgt caccgtcacc 540tgggaggtgg
atggcaccac ccaaacaact ggcatcgaga acagtaaaac accgcagaat 600tctgcagatt
gtacctacaa cctcagcagc actctgacac tgaccagcac acagtacaac 660agccacaaag
agtacacctg caaggtgacc cagggcacga cctcagtcgt ccagagcttc 720aataggggtg
actgttagag tgagagcggc cgc
7531231421DNAArtificial SequenceSynthetic Construct 123aagcttgtac
ccttcaccat ggagactggg ctgcgctggc ttctcctggt cgctgtgctc 60aaaggtgtcc
agtgtcagtc gttggaggag tccgggggtc gcctggtcac gcctgggaca 120cccctgacac
tcacctgcac cgtctctgga ttctccctca gtcgcaatgc aataaactgg 180gtccgccagg
ctccagggaa ggggctggaa tacatcggaa tcattggtag tagtggtgtc 240acatactacg
cgagctgggc aaaaggccga ttcaccatct ccagaacctc gaccacggtg 300gatctgaaaa
tcaccagtcc gacaaccgag gacacggcca cctatttttg tgccagaaat 360ctttatactg
gtggtagtaa tgataacttg tggggcccag gcaccctggt caccgtctcc 420tcagggcaac
ctaaggctcc atcagtcttc ccactggccc cctgctgcgg ggacacaccc 480agctccacgg
tgaccctggg ctgcctggtc aaagggtacc tcccggagcc agtgaccgtg 540acctggaact
cgggcaccct caccaatggg gtacgcacct tcccgtccgt ccggcagtcc 600tcaggcctct
actcgctgag cagcgtggtg agcgtgacct caagcagcca gcccgtcacc 660tgcaacgtgg
cccacccagc caccaacacc aaagtggaca agaccgttgc gccctcgaca 720tgcagcaagc
ccacgtgccc accccctgaa ctcctggggg gaccgtctgt cttcatcttc 780cccccaaaac
ccaaggacac cctcatgatc tcacgcaccc ccgaggtcac atgcgtggtg 840gtggacgtga
gccaggatga ccccgaggtg cagttcacat ggtacataaa caacgagcag 900gtgcgcaccg
cccggccgcc gctacgggag cagcagttca acagcacgat ccgcgtggtc 960agcaccctcc
ccatcgcgca ccaggactgg ctgaggggca aggagttcaa gtgcaaagtc 1020cacaacaagg
cactcccggc ccccatcgag aaaaccatct ccaaagccag agggcagccc 1080ctggagccga
aggtctacac catgggccct ccccgggagg agctgagcag caggtcggtc 1140agcctgacct
gcatgatcaa cggcttctac ccttccgaca tctcggtgga gtgggagaag 1200aacgggaagg
cagaggacaa ctacaagacc acgccggccg tgctggacag cgacggctcc 1260tacttcctct
acagcaagct ctcagtgccc acgagtgagt ggcagcgggg cgacgtcttc 1320acctgctccg
tgatgcacga ggccttgcac aaccactaca cgcagaagtc catctcccgc 1380tctccgggta
aatgagcgct gtgccggcga gctgcggccg c
14211241415DNAArtificial SequenceSynthetic Construct 124aagcttgtac
ccttcaccat ggagactggg ctgcgctggc ttctcctggt cgctgtgctc 60aaaggtgtcc
agtgtcagtc ggtggaggag tccgggggtc gcctggtcac gcctgggaca 120cccctgacac
tcacctgcaa agcctctgga ttctccctca gtgactctag agtgagctgg 180gtccgccagg
ctccagggaa ggggctggaa tggatcggaa tcgttggcat tggttggaat 240atataccacg
cgaactgggc gaaaggccga ttcaccatct ccaaaacgtc gtcgaccacg 300gtggatttga
aaatcaccag tccgacagtc gaggacacgg ccacctattt ctgtgccaga 360ggtctgggtg
gtggtactgt catctggggc ccaggcaccc tggtcaccgt ctccttaggg 420caacctaagg
ctccatcagt cttcccactg gccccctgct gcggggacac acccagctcc 480acggtgaccc
tgggctgcct ggtcaaaggg tacctcccgg agccagtgac cgtgacctgg 540aactcgggca
ccctcaccaa tggggtacgc accttcccgt ccgtccggca gtcctcaggc 600ctctactcgc
tgagcagcgt ggtgagcgtg acctcaagca gccagcccgt cacctgcaac 660gtggcccacc
cagccaccaa caccaaagtg gacaagaccg ttgcgccctc gacatgcagc 720aagcccacgt
gcccaccccc tgaactcctg gggggaccgt ctgtcttcat cttcccccca 780aaacccaagg
acaccctcat gatctcacgc acccccgagg tcacatgcgt ggtggtggac 840gtgagccagg
atgaccccga ggtgcagttc acatggtaca taaacaacga gcaggtgcgc 900accgcccggc
cgccgctacg ggagcagcag ttcaacagca cgatccgcgt ggtcagcacc 960ctccccatcg
cgcaccagga ctggctgagg ggcaaggagt tcaagtgcaa agtccacaac 1020aaggcactcc
cggcccccat cgagaaaacc atctccaaag ccagagggca gcccctggag 1080ccgaaggtct
acaccatggg ccctccccgg gaggagctga gcagcaggtc ggtcagcctg 1140acctgcatga
tcaacggctt ctacccttcc gacatctcgg tggagtggga gaagaacggg 1200aaggcagagg
acaactacaa gaccacgccg gccgtgctgg acagcgacgg ctcctacttc 1260ctctacagca
agctctcagt gcccacgagt gagtggcagc ggggcgacgt cttcacctgc 1320tccgtgatgc
acgaggcctt gcacaaccac tacacgcaga agtccatctc ccgctctccg 1380ggtaaatgag
cgctgtgccg gcgagctgcg gccgc 1415
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