VACCINES AGAINST AN ONCOGENIC ISOFORM OF ESR1 AND METHODS OF USING THE SAME

Abstract

Methods of reducing the likelihood of a cancer or precancer developing resistance to a cancer therapeutic or prevention agent are provided herein. The methods include administering the cancer therapeutic or prevention agent and a vaccine comprising a polynucleotide encoding a polypeptide whose expression or activation is correlated with development of resistance of the cancer or precancer to the cancer therapeutic or prevention agent to a subject. The vaccine may include a polynucleotide encoding an ESR1 polypeptide or a truncation, deletion or substitution mutant thereof. Methods of using the vaccine including the polynucleotide encoding the ESR1 polypeptide to treat a cancer or precancer are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of priority of U.S. Provisional Patent Application No. 62/021,586, filed Jul. 7, 2014, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0003] A Sequence Listing accompanies this application and is incorporated herein by reference in its entirety. The Sequence Listing was filed with the application as a text file on Jul. 7, 2015.

INTRODUCTION

[0004] This application relates to a cancer vaccine against ESR1, specifically a vaccine against ESR1 isoform antigens that are expressed on cancer cells or in response to development of resistance to a therapeutic intervention to cancer (or pre-cancers). Methods of using the vaccines are also provided.

[0005] Cancer vaccines target antigens expressed by tumors, but application of these vaccines has not been as effective as once hoped due to induction of immune tolerance by chronic overexpression of the targeted protein in the absence of co-stimulatory molecules and the induction of an immunomodulatory environment. Preventative cancer vaccines may be more promising, but cancers are highly variable, with multiple genetic changes, but few truly universal changes. Thus, it is difficult to predict what antigens will be overexpressed on any specific cancer or whether an individual should be vaccinated and if so, with what antigens. In contrast, a strategy is proposed here in which vaccination against the antigen(s) that will predictably be overexpressed in response to a therapy, but prior to that antigen's over-expression by the cancer cells is used to induce a robust anti-cancer immune response.

SUMMARY

[0006] Provided herein is a mechanism of revolutionizing cancer therapy or prevention by preventing the development of resistance to cancer therapeutic or cancer prevention agents by identifying which antigens are likely to be expressed in a cancer or precancer in response to treatment with a cancer therapeutic or prevention agent and thus which antigens may be targeted with a vaccine in patients.

[0007] A vaccine targeting a specific antigen involved in a resistance mechanism, namely ESR1, and methods of using the vaccine are provided. In one aspect, the vaccine includes a polynucleotide encoding an ESR1 polypeptide, a mutant of an ESR1 polypeptide, or a portion of an ESR1 polypeptide. For example, ESR1 polypeptides of SEQ ID NO: 1-3 or 5 or portions thereof may be included in a vaccine as detailed below. SEQ ID NO: 1-3 and 5 each provide single amino acid substitution mutants of ESR1. SEQ ID NO: 1 is an ESR polypeptide with an Y537N mutation. SEQ ID NO: 2 is an ESR1 polypeptide with a Y537S mutation. SEQ ID NO: 3 is an ESR1 polypeptide with a D538G mutation. SEQ ID NO: 5 is an ESR1 polypeptide with a K303R mutation.

[0008] In another aspect, methods of treating a cancer or precancer or reducing the likelihood of the cancer or precancer to develop resistance to a cancer therapeutic or prevention agent by administering the vaccine provided herein to a subject with cancer or precancer are provided. The vaccine may be administered before, concurrently with or after administration of the cancer therapeutic or prevention agent. The vaccine may also be administered before, concurrently with or after administration of checkpoint inhibitory immunomodulatory agents such as antagonistic antibodies specific for CTLA-4 or PD1.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 is a graph showing that ESR1 mutants can confer ER Signaling in the absence of estrogen stimulation. 293T cells stably expressing the indicated genes were transfected with an ERE luciferase reporter (180 ng), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

[0010] FIG. 2 is a graph showing analysis of ESR1-Y537N mutant signaling. 293T cells stably expressing the indicated genes were reverse transfected with 45 different pathway reporter luciferase vectors (180 ng/well), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

[0011] FIG. 3 is a graph showing analysis of ESR1-K303R mutant signaling. 293T cells stably expressing the indicated genes were reverse transfected with 45 different pathway reporter luciferase vectors (180 ng/well), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

[0012] FIG. 4 is a graph showing ESR1 PR signaling with different mutants. 293T cells stably expressing the indicated genes were transfected with an PR luciferase reporter (180 ng), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

[0013] FIG. 5 is a graph showing ESR1 RAR signaling with different mutants. 293T cells stably expressing the indicated genes were transfected with an RAR luciferase reporter (180 ng), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

[0014] FIG. 6 is a set of figures showing that ESR1-mutants can confer estrogen-independent growth in ER+ breast cancer in vivo. Stably infected MCF-7 cells were injected into the flank of SCID-Beige male mice (or female where indicated) at a concentration of 1M cells in 100 .mu.l of PBS at Day 0. In female control group, a 17B-Estradiol pellet (60 day release, Innovative Research of America, Sarasota Fla.) was also implanted. Tumor growth was then monitored as indicated by caliper measurement (N=5 or 10 per group, bars represent SE) and is shown in the graph (FIG. 6A) and reported in the table (FIG. 6B). The photograph at the bottom right of the figure (FIG. 6C) is from a previous experiment measuring the growth of MCF-7-ESR1-Y537N cells in female mice supplemented with 17B-Estradiol pellets (top) versus male mice without supplementation (bottom).

[0015] FIG. 7 is a graph showing ESR1 ERE signaling in murine mammary cells with different mutants. MM3MG cells stably expressing the indicated genes were transfected with an ERE luciferase reporter (180 ng), along with a LacZ control (20 ng) using FugeneHD. At 24 hours post-transfection, cells were split and plated into 96 well plates (20,000 cells per well) where they were treated with or without 17B-Estradiol (20 nM) as indicated. 24 hours post-treatment, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

[0016] FIG. 8 is a graph showing that ESR1 mutants in murine mammary cells do not confer proliferative advantage. MM3MG cells stably expressing the indicated genes were plated in 96-well plates (5,000 per well) and assessed at 4 days post-plating by MTT Assay (N=12, bars indicate SD).

[0017] FIG. 9 is a set of figures showing that ESR1 mutants in murine mammary cells can confer an advantage in anchorage-independent growth. MM3MG cells stably expressing the indicated genes were plated in 12-well dishes plates (2,500 per well) and assessed at 3 weeks days post-plating at 4.times. and 10.times. magnification (N=4, bars indicate SD). FIG. 9A is a graph showing the number of colonies per well for cells expressing the indicated ESR1 protein and either exposed to eatrogen or not. FIG. 9B is a set of photographs showing anchorage independent cell growth of the indicated cells.

[0018] FIG. 10 is a graph showing ESR1 mutants in murine mammary cells can confer a proliferative advantage in vivo. MM3MG cells stably expressing the indicated genes were implanted subcutaneously into SCID-Beige mice (100,000 per mouse in PBS) at days 0. Tumor growth was measured by calipers at the indicated days (N=5, bars represent SE).

[0019] FIG. 11 is a graph showing that the adenoviral vaccines targeting ESR1 elicit significant T-cell responses against ESR1-specific epitopes. C57 mice were vaccinated using the indicated adenoviral vectors (2.6E10 viral particles per mouse via footpad) and sacrificed at 2 wpi. ELISPOT assays were then performed using 500 k splenocytes per well against the indicated antigen stimuli (N=5, bars represent SD).

[0020] FIG. 12 is a graph showing that the adenoviral vaccines targeting ESR1 elicit significant B-cell responses against ESR-specific epitopes. C57 mice were vaccinated using the indicated adenoviral vectors (2.6E10 viral particles per mouse via footpad) and sacrificed at 2 wpi. ELISA assays were then performed using ESR1 coated plates (Thermo Fisher, 10 g/ml) and using an anti-mouse lgG-HRP secondary antibody (CST, 1:1000 dilution) to detect ESR1-specific IgG antibodies. (N=5, bars represent SD).

[0021] FIG. 13 is a graph showing that targeted vaccination against ESR1 mutants suppresses the growth of ESR1-mutant expressing cells. BALB/c mice were vaccinated using the indicated adenoviral vectors (2.6E10 viral particles per mouse via footpad) and MM3MG cells stably expressing the indicated genes were implanted at 2 wpi (100,000 per mouse in PBS, indicated at day 0). Tumor growth was measured by calipers at the indicated days (N=5, bars represent SE).

[0022] FIG. 14 is a graph showing that the adenoviral vaccines targeting ESR1 elicit T-cell responses against ESR1-specific epitopes in tumor-bearing mice. BALB/c mice were vaccinated using the indicated adenoviral vectors (2.6E10 viral particles per mouse via footpad) and MM3MG cells stably expressing the indicated genes were implanted at 2 wpi (100,000 per mouse in PBS, indicated at day 0). Tumor growth was measured by calipers at the indicated days (N=5, bars represent SE). ELISPOT assays were then performed using 500 k splenocytes per well against the indicated antigen stimuli (N=5, bars represent SD).

DETAILED DESCRIPTION

[0023] Approximately 70% of all breast cancers are classified as estrogen receptor positive (ER+); dependent upon constitutive estrogen receptor signaling Although different classes of endocrine (anti-estrogen) therapies (including selective estrogen receptor modulators (SERMS), downregulators, and aromatase inhibitors (AIs)) are effective treatments for these cancers in adjuvant settings, approximately 50% of women will eventually relapse and die from metastatic ER+ disease.sup.7-9. Thus, despite the advent of newer therapies (such as AIs) there remains an unrelenting rate of recurrence in ER+ breast cancer, particularly in cases where metastasis has occurred.sup.10-12. Significantly, all patients that develop metastatic ER+ disease will progress to an endocrine therapy resistant disease. At this stage, there is no cure for ER+ breast cancer.

[0024] Because compensatory mechanisms appear to account for resistance that develops in a significant percentage of anti-estrogen treated patients, we propose a novel approach that has the potential to target critical driver mutations for the lifetime of the patient. Provided herein is specifically targeted immunotherapy directed toward specific resistance drivers that are predictably evoked by compensatory resistance mechanisms.

[0025] As a novel alternative to vaccines targeting well established tumor antigens, we hypothesized that the antigen-specific immune non-responsiveness to conventional tumor-associated antigens may be avoided by targeting tumor antigens that are induced after exposure to a cancer therapeutic or prevention agent as a mechanism of developing therapeutic resistance.

[0026] Although there may be many potential antigens overexpressed in response to a cancer therapeutic or prevention agent, those antigens that are likely critical components of specific therapeutic resistance mechanisms would be attractive targets, as immunologic ablation of clones expressing such antigens should eliminate the clinical recurrence of therapy resistant tumor cells. One such antigen thought to be essential to therapeutic resistance is the estrogen receptor. ESR1. As proof of this concept in ER+ breast cancer, we have chosen to focus this approach on the recently uncovered resistance mutations to Estrogen Receptor alpha (ESR1).

[0027] We recently demonstrated that polyclonal antibodies induced by vaccination against receptors such as HER2 and HER3 can mediate profound receptor internalization and degradation, providing a therapeutic effect in vitro and in vivo (Ren et al., Breast Cancer Research 2012 14: R89 and International Patent Application No. WO 2013/110030, both of which are incorporated herein by reference in their entireties).

[0028] A vaccine composed of single or multiple forms of the ESR1 gene (Estrogen Receptor alpha) encoded by a platform that would elicit an immune response to the wild type or mutated epitopes of ESR1 is provided herein. In one embodiment, vaccines comprising one or more (or all) or portions of one or more (or all) of SEQ ID NOs: 1-3 and SEQ ID NO: 5 are provided. In the Examples, each vaccine contained a single ESR1 mutant, but more than one mutant can be included in a single vaccine. While others have utilized different approaches to target wild-type ESR1 through vaccination, our approach would selectively target forms of this gene that enable endocrine therapy resistance in tumors. i.e. mutant forms of ESR1 that allow the cancer cells to escape a therapeutic agent and continue growth. As such, we would expect that targeting these forms would prevent their emergence and would effectively prevent the development of resistance to endocrine therapies in ER+ breast cancer as well as other endocrine dependent cancers. Additionally, selectively targeting this specific mutant form of ESR1 would allow for effective tumor-specific anti-cancer activity mediated through immune targeting.

[0029] This invention would optimally be utilized through the inclusion of antigens encoded by the wild type and/or mutant forms of ESR1 gene (Y537N, Y537S, D538G, K303R and others), optimized forms of this gene (truncated, inactivated or otherwise), or specific combinations of peptide/epitopes of this gene in different immune stimulatory vector systems. The mutant forms of ESR1 gene (Y537N, Y537S, D538G, K303R) are provided as SEQ ID NOs; 1-3 and 5, respectively. Portions of these polypeptides may also be included in the vaccine. Suitably the portion included in the vaccine includes the mutation at the indicated amino acid. The portion included in the vaccine may include the mutation at only one of 537, 538 or 303 or may include small peptide epitopes comprising two of more of these mutations. The vaccine may include further peptides with additional mutations in the ESR1 protein in addition to those identified herein. The B and T cell epitopes being recognized after vaccination in the examples have not been identified, but those of skill in the art would expect the epitopes to be 6, 8, 10, 12, 14, 16, 18 or 20 amino acids in length. The Examples do suggest that the epitope includes the mutation at 537 for the vaccine containing SEQ ID NO:1 as the immune response generated after vaccination with the vaccine comprising SEQ ID NO:1 did not recognize wild-type ESR1. The vaccines used in the Examples encompass larger polypeptides, but vaccines may include smaller portions of the ESR1 polypeptides than those provided herein. Suitably the vaccines include the region flanking the mutations at amino acid 537, 538 or 303 of the sequences and include at least 8, 10, 12, 14, 16, 18, 20 or more amino acids.

[0030] A polynucleotide encoding a polypeptide of SEQ ID NO: 1-3 or 5 or a portion thereof may be encompassed in a vaccine vector. Suitable vaccine vectors include, but are not limited to viral vectors such as adenoviral, fowlpox, vaccinia, VEE, etc., DNA-based vaccination vectors, or protein/peptide vaccination strategies. Liposomes or bacterial vaccine vectors may also be suitable. This immunotherapeutic platform could be used prior to the development of cancer types prior to the development of endocrine resistance, used in front line or adjuvant settings as a treatment for these cancers, and also as a preventative measure to prohibit the development and evolution of this signaling pathway as a resistance pathway.

[0031] The vaccines or vaccine vectors may include polynucleotides encoding additional polypeptides, such as HER3, HER2 or polypeptides of either of these comprising mutations such as those provided in SEQ ID NOs: 6-10 or any of the epitopes provided in International Publication No. WO2013/110030, which is incorporated herein by reference in its entirety. The vaccines or vaccine vectors may also include or be administered in conjunction with a checkpoint inhibitory immunomodulatory agent. The checkpoint inhibitory immunomodulatory agent may be an antibody antagonistic for CTLA-4 or PD1. In the Examples a PD1 antibody obtained from BioXCell called RMP1-14 and a CTLA-4 antibody from BioXCell called 9D9 were used. Other similar antibodies are commercially available or in clinical trials such as ipilimumab and nivolumab.

[0032] This would be easily distinguished from our and other prior approaches targeting wild-type ESR1 as the mutations to different portions of this gene render them insensitive to endocrine-targeted therapies with enhanced oncogenic potential. As such, vaccines targeting these mutant forms may elicit a different epitope repertoire for immune targeting and potentially a more significant anti-tumor effect by specifically targeting the development of endocrine resistance and specifically preventing it through immunoselective pressure.

[0033] Generation of resistance to cancer therapeutic or prevention agents is a common problem in the treatment of cancer or precancer and in several cases the mechanism of resistance to the therapeutic agent is known. Resistance is often the result of changes in gene expression (over-expression or blocked expression of a protein), change in the gene by mutation, or altered sequences by altered splicing or translocation or altered activation of a protein in the cells (over-activation or blocked activation of a protein).

[0034] In those cases where over-expression or over-activation of a protein, or a new sequence in the protein is responsible for increasing the resistance of the cancer or precancer cells to the therapeutic or prevention agent, we report a method for reducing the likelihood that the cancer or precancer will develop resistance to the cancer therapeutic or prevention agent. As used herein, resistance to a cancer therapeutic or prevention agent indicates that the cancer therapeutic or prevention agent is not as effective at inhibiting the growth of, or killing, cancer or precancer cells in response to the cancer therapeutic or prevention agent. The method may even block the development of resistance to the cancer therapeutic or prevention agent or may reverse resistance to the cancer therapeutic or prevention agent after it has developed. The methods include administering the cancer therapeutic or prevention agent and administering a vaccine to the subject in need of treatment for a cancer. The vaccine comprises a polynucleotide encoding a polypeptide whose expression or activation is correlated with or results in development of resistance of the cancer or precancer to the cancer therapeutic or prevention agent. The vaccines provided herein include an ESR1 polypeptide or a polynucleotide encoding an ESR1 polypeptide such as the polypeptide of SEQ ID NO: 2, 4, or 6.

[0035] The vaccine may be administered before, during or after treatment with a cancer therapeutic or prevention agent or may be administered simultaneously with the cancer therapeutic or prevention agent. The administration of the vaccine and the cancer therapeutic or prevention agent to the subject reduces the likelihood that the subject's cancer or precancer will develop resistance to the therapeutic or prevention agent as compared to a control subject with a similar cancer or precancer not administered the vaccine or as compared to the general likelihood of a population of subjects having the cancer or precancer. In some embodiments, the cancer or precancer in individuals administered both the vaccine and the therapeutic or prevention agent does not develop resistance to the cancer therapeutic or prevention agent and is treated. Alternatively, the growth of the cancer or precancer may be inhibited or the growth rate reduced. The administration of the vaccine and cancer therapeutic or prevention agent may also reverse resistance to the cancer therapeutic or prevention agent if the cancer or precancer is already resistant to the cancer therapeutic or prevention agent. In some embodiments, administration of the vaccine is sufficient to treat the cancer or inhibit the growth or kill the cancer. In other embodiments, the vaccine must be administered in conjunction with the cancer therapeutic or prevention agent or prior to development of resistance to the cancer therapeutic or prevention agent by the cancer.

[0036] The vaccine may include a polynucleotide encoding an ESR1 polypeptide. Three point mutations (4 mutant forms) of ESR1 associated with resistance to cancer therapeutic agents are provided as SEQ ID NOs: 1-3 and 5. The vaccine may comprise full-length ESR1 or portions thereof. For example, the vaccine may comprise only the epitopes identified in the examples or peptides comprising the mutations or deletions associated with resistance. Suitably the vaccine is capable of eliciting an immune response to ESR1 in a subject administered the vaccine. The immune response may be a B cell or T cell response. Suitably the immune response includes an antibody response directed to ESR1. The immune response may be a polyclonal antibody response in which multiple epitopes of ESR1 are recognized by antibodies. The immune response may recognize an epitope including the mutations such that after immunization the wild-type protein is not recognized or not recognized as strongly as the mutant form.

[0037] ESR1, as shown in SEQ ID NOs: 1-3 and 5, comprises mutations as indicated that lead to endocrine resistance. The mutations result in a unique sequence in the peptide and epitopes spanning these mutations can be identified and antibodies generated using the vaccines described herein. Those of skill in the an will appreciate that a vaccine including polynucleotides encoding only portions of full-length ESR1, i.e. antigenic epitopes, may be used in the vaccines described herein. Portions of the ESR1 including the mutation sites can be included in the vaccine. We have the following isolated ESR and ESR related peptides presented by MC molecules on tumor cells.

TABLE-US-00001 TABLE 1 T cell epitopes derived from estrogen receptor related proteins: MCF10 (SEQ ID NO:) IQGNELEPL A2 Estrogen receptor ESR1 (11) MCF7 FMVLQVIKF A24 nuclear receptor (12) subfamily 1 group I member 3 isoform 15 LEMLEAKV non A2/A24 estrogen-related receptor (13) beta MDA EVFLPQRA A2 estrogen-related receptor (14) beta IFLNTEVSL A24 estrogen receptor (15) coactivator LTAEETDKI A2/A24 estrogen receptor (16) coactivator LTSSSIDPGL A2 estrogen receptor binding (17) protein variant MLKHKRPLA A2/A24 estrogen receptor alpha (18) splice variant, partial TIVSLDAARR A2 estrogen-responsive B box (19) protein KGDEEKENN non A2/A24 estrogen receptor-related (20) protein LCVKAMILL non A2/A24 estrogen receptor 2 (21) (ER beta) variant MNQKLSPFM non A2/A24 estrogen sulfotransferase (22) RYKKLKVE non A2/A24 estrogen-related receptor (23) beta SKAKSLTDPS non A2/A24 estrogen receptor binding (24) protein variant WFGIKAPE non A2/A24 estrogen receptor binding (25) protein variant

Any of these polypeptides individually or in combination may be used as ESR1 polypeptides in the vaccine described herein.

[0038] The vaccine may include a vaccine vector. The vaccine vector may be a bacterial, yeast, viral or liposomal vaccine vector. The vaccine may be a DNA vaccine as well and not include a vaccine vector. The vaccine vector may be an adenovirus, adeno-associated virus, fowlpox, vaccinia, viral equine encephalitis virus, venezuelan equine encephalitis virus or other viral vaccine vectors. One method for generating adenovirus vectors is provided in Luo et al., Nature Protocols, (2007) 2: 1236-1247, which is incorporated herein by reference. The vaccine vector may contain the ESR1 polynucleotide or portions thereof. The vaccine vector may contain the ESR1 polypeptide or portions thereof. The vaccine vector may express the ESR1 polypeptide or portions thereof. ESR1 polypeptide or portions thereof may be expressed on the surface or interior of the vaccine vector. ESR1 polynucleotide or portions thereof may be carried within the vaccine vector and the ESR1 polypeptide or portions thereof may be expressed only after vaccination. ESR1 polypeptides or portions thereof may be expressed as a fusion protein or in conjunction with adjuvants or other immunostimulatory molecules to further enhance the immune response to the polypeptide.

[0039] Methods of treating a cancer or precancer, or of reducing the likelihood of the cancer or precancer developing resistance to a cancer therapeutic or prevention agent, are also provided. The methods include administering the vaccine as described above to a subject having cancer or precancer. The subject may be any mammal, suitably a human, domesticated animal such as a dog or cat, or a mouse or rat. A cancer therapeutic or prevention agent may be administered concurrently with, before or after administration of the vaccine.

[0040] The cancer therapeutic or prevention agents may be any agent capable of treating the cancer or inhibiting growth of cancer cells. Suitable agents include those which target HER2, HER1/EGFR, HER3, estrogen receptor or IGF1R. The therapeutic agent may be trastuzumab, lapatinib, pertuzumab or another HER2 or estrogen receptor targeting therapeutic agent or it may be an EGFR targeting therapeutic agent such as cetuximab or erlotanib, or it may be an anti-estrogen, or an agent that prevents estrogen synthesis such as an aromatase inhibitor. We have previously demonstrated that a HER3 vaccine can treat a HER2 positive cancer when used in combination with a therapeutic agent targeting HER2. An ESR1 vaccine should work similarly and the mutations provide unique sites for vaccination to differentiate cancer or precancer cells from normal cells. Cancer cells often develop resistance to therapeutic agents. Addition of vaccination with an ESR1 vaccine or passively transferred polyclonal antibodies specific for ESR1 will result in blocking resistance, inhibit cancer cell growth and result in treatment of the cancer.

[0041] Suitably the vaccinated subject develops an immune response to the mutated form of ESR1 in response to administration of the vaccine. The immune response may be an antibody or T cell immune response. For example the immune response may include antibody-dependent cellular cytotoxicity, polyclonal antibody response, complement dependent cellular cytotoxicity, cellular cytotoxicity, disruption of ligand binding, disruption of dimerization, mimicking ligand binding causing internalization of ESR1, or degradation of ESR1. The immune response may comprise an antibody response directed to at least a portion of ESR1, suitably a portion including the mutations. The immune response may be specific for a T cell or B cell epitope flanking or encompassing the mutations in SEQ ID NO: 1-3 or 5 or regions flanking the mutations in ESR1.

[0042] Reduction of the development of resistance can be measured in several ways. The resistance of the vaccinated subject may be compared to a similar subject that was not vaccinated as in the Examples. Alternatively, the reduction may be measured based on statistics generated regarding the likelihood of an individual being treated with the therapeutic agent to develop resistance versus that of individuals treated with the therapeutic agent and vaccinated with ESR1. The reduction in the likelihood of resistance of the cancer may also be measured by measuring the level of ESR1 expression on the surface of cancer cells. ESR1 expression is reduced on cancer cells after effective administration of the vaccine. The effectiveness of the vaccine in treating the cancer or reducing the likelihood of resistance can be measured by tracking the growth of the tumor or the growth rate of the tumor or cancer cells. A decrease in tumor size or in the rate of tumor growth is indicative of treatment of the cancer.

[0043] The cancer may be selected from any cancer capable of developing resistance to a therapeutic agent by increasing expression or activation of a protein by the cancer cells. In particular the cancer may be any cancer capable of developing resistance to a therapeutic agent which targets a HER family tyrosine kinase, suitably HER2, HER3, or EGFR or the estrogen receptor, suitably anti-estrogens. The cancer may develop resistance by increasing the expression of ESR1, mutating ESR1 or deleting a portion of ESR1 to avoid susceptibility to the therapeutic agent. Suitably the cancers are selected from breast, prostate, lung, ovarian, colon, rectal, pancreas, bladder, head and neck or liver cancers or precancers. The resistance may be due to a single or multiple changes, and the vaccine can target one or more of these changes, and/or include multiple antigens likely found in resistance cells, but not necessarily in all resistance cells.

[0044] Treating cancer includes, but is not limited to, reducing the number of cancer cells or the size of a tumor in the subject, reducing progression of a cancer to a more aggressive form (i.e. maintaining the cancer in a form that is susceptible to a therapeutic agent), reducing proliferation of cancer cells or reducing the speed of tumor growth, killing of cancer cells, reducing metastasis of cancer cells or reducing the likelihood of recurrence of a cancer in a subject. Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with cancer or at risk of developing cancer or facing a cancer recurrence. Treatment includes improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay in the onset of symptoms or slowing the progression of symptoms, etc.

[0045] Co-administration, or administration of more than one composition (i.e. a vaccine and a therapeutic agent) to a subject, indicates that the compositions may be administered in any order, at the same time or as part of a unitary composition. The two compositions may be administered such that one is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more.

[0046] An effective amount or a therapeutically effective amount as used herein means the amount of a composition that, when administered to a subject for treating a state, disorder or condition is sufficient to effect a treatment (as defined above). The therapeutically effective amount will vary depending on the compound, formulation or composition, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated.

[0047] The compositions (i.e. the vaccines and the therapeutic agents or checkpoint inhibitory agents) described herein may be administered by any means known to those skilled in the art, including, but not limited to, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, or transmucosal absorption. Thus the compositions may be formulated as an ingestable, injectable, topical or suppository formulation. The compositions may also be delivered with in a liposomal or time-release vehicle. Administration of the compositions to a subject in accordance with the invention appears to exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, administration of larger quantities of the compositions is expected to achieve increased beneficial biological effects than administration of a smaller amount. Moreover, efficacy is also contemplated at dosages below the level at which toxicity is seen.

[0048] It will be appreciated that the specific dosage administered in any given case will be adjusted in accordance with the composition or compositions being administered, the disease to be treated or inhibited, the condition of the subject, and other relevant medical factors that may modify the activity of the compositions or the response of the subject, as is well known by those skilled in the art. For example, the specific dose for a particular subject depends on age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given patient can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the compositions described herein and of a known agent, such as by means of an appropriate conventional pharmacological or prophylactic protocol.

[0049] The maximal dosage for a subject is the highest dosage that does not cause undesirable or intolerable side effects. The number of variables in regard to an individual prophylactic or treatment regimen is large, and a considerable range of doses is expected. The route of administration will also impact the dosage requirements. It is anticipated that dosages of the compositions will reduce the growth of the cancer at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more as compared to no treatment or treatment with only the therapeutic agent. It is specifically contemplated that pharmaceutical preparations and compositions may palliate, block further growth or alleviate symptoms associated with the cancer without providing a cure, or, in some embodiments, may be used to cure the cancer and rid the subject of the disease.

[0050] The effective dosage amounts described herein refer to total amounts administered, that is, if more than one composition is administered, the effective dosage amounts correspond to the total amount administered. The compositions can be administered as a single dose or as divided doses. For example, the composition may be administered two or more times separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days, three days, four days, one week, two weeks, or by three or more weeks.

[0051] The vaccine vector may be administered one time or more than one time to the subject to effectively boost the immune response against ESR1. If the vaccine is provided as a vaccine vector, the vaccine vector may be administered based on the number of particles delivered to the subject (i.e. plaque forming units or colony forming units). The subject may be administered 10.sup.12, 10.sup.11, 10.sup.10, 10.sup.9, 10.sup.8, 10.sup.7 or 10.sup.6 particles.

[0052] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms"including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as "including," "comprising," or "having" certain elements are also contemplated as "consisting essentially of" and "consisting of" those certain elements.

[0053] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word "about" to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0054] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.

[0055] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

Examples

Materials and Methods

[0056] Viral Vectors: Mutations in the ESR plasmid were generated by site-directed mutatgenesis of a pENTR221-ESR1 plasmid obtained from the Orfeome (Dharmacon) to create ESR1 mutants Y537N, Y537S, D538G, and K303R. See SEQ ID Nos: 1-3 and 5, respectively. These genes were cloned into adenoviral vectors, which were then generated as previously described.sup.1. These genes were also cloned into lentiviral vectors (LX301 from Addgene) and used to generated cell lines with stable expression of these genes as previously described.sup.2.

[0057] Cell Lines: Breast epithelial cell lines MM3MG, NMuMG, and MCF-7 were obtained from the American Tissue Culture Collection (ATCC), and were maintained according to ATCC recommendations. These lines were tested for Mycoplasma and DNA fingerprinted at the Duke Cell Culture Facility.

[0058] In Vitro Assays: Proliferation of stable cells was determined by MTT assay using 5,000 cells/well over the course of 3 days (against control counterparts) in 96-well plates. MTT growth assessments were done using a Bio-Rad plate reader after cell solubilization in DMSO. Soft agar assays for stable expression were done as described.sup.3. Briefly, 50,000 cells/well were plated in 0.3% soft agar (on a base of 0.6% soft agar) and allowed to grow for a period of 2 wk in DMEM with 10% FBS. At the end of this time, colonies of >15 cells were counted and scored. In experiments using inducible expression systems, Dox was added to the media at a concentration of 2 .mu.g/ml (replaced weekly). Wound Scratch Assays were performed using p1000 tips, washing wounded plates with PBS (2.times.) and applying media before staining with Crystal Violet at 16 hours post-wounding. Pictures were taken using an Olympus 1X73 using a 10.times. magnification objective. Luciferase Assays were performed by co-transfecting an luciferase reporter plasmids (SABiosciences) along with a CAG-LacZ control (Addgene) into modified 293T cells in estrogen-free conditions (phenol-red free media with charcoal stripped FBS) and assessing pathways signaling at 24 or 48hpt using standard techniques with Luminometer (GloMax 96-well, Promega, Madison Wis.).

[0059] Animal Experiments: Experiments using BALB/c and SCID-Beige mice (obtained from Jackson Labs) were done in accordance with Duke Institutional Animal Care and Use Committee-approved protocols. For tumor vaccine experiments, BALB/c mice (Jackson Labs) were implanted with MM3MG modified cellsR2d16 tumors and genotyped by PCR as previously described.sup.4. In immune competent (BALB/c) and immuno-deficient animals (SCID-beige), stable cells were injected s.c. into the flank of SCID-beige mice (at indicated cells/animal) and measured as indicated. Tumor measurements were made using calipers and volumes calculated using the formula [v=width.times.width.times.(length/2)] whereas statistical differences were calculated using a mixed effects regression model using autoregressive covariance.

[0060] ELISPOT and ELISA Assays: Immunogenicity experiments involved footpad injection of Ad-ESR-WT, ESR1-mutant, and Ad-GFP vectors (2.6.times.10.sup.10 particles/mouse) in BALB/c animals. Fourteen days post-injection, mice were euthanized and splenocytes and sera were collected for analysis. IFN-.gamma. ELISPOT assays (Mabtech Inc.) were done according to the manufacturer's instructions using overlapping ESR1 peptide mixes (2.6 .mu.g/mL; BD Biosciences) as stimulating antigens and HIV-irrelevant overlapping peptide mixes as negative controls (BD Biosciences). Phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (1 .mu.g/mL) served as a positive control for splenocyte responsiveness. Antibodies were assessed using a sandwich-based ELISA method using ESR1 protein (10 ug/ml, Thermo-Fisher) as previously described using LacZ.sup.5.

Results:

[0061] To initially validate the importance of the identified ESR1 mutations, we first investigated their ability to elicit canonical estrogen-dependent signaling in the absence of exogenous estrogen stimulation. To test differences in ESR1 estrogen-dependent signaling, we generated 293T cells (which do not express detectable levels of ESR1) expressing ESR-WT, Y537N, Y537S, D538G, and K303R. Using a highly sensitive canonical ERE-pathway luciferase-based signaling reporter system, we then determined how estrogen-free and estrogen-induced ERE pathways were affected by ESR1-WT or mutant expression. We found that ERE signaling was strongly activated in ESR1-WT expressing cells when stimulated with estrogen in comparison to ESR1-WT expressing non-stimulated cells or control cells stimulated with estrogen (FIG. 1). More importantly, all mutant forms of ESR1 strongly induced the ERE pathway in the absence of estrogen (FIG. 1). To get a broader view of potential pathways affected by these mutants, we utilized a high throughput 45-pathways reporter array (SABiosciences) for both estrogen receptor mutation classes (represented by ESR1-Y537S and K303R). These experiments revealed that both classes of estrogen receptor mutants elicited ERE signaling, but also PR and RAR signaling pathways (FIGS. 2 and 3). Having identified the three critical pathways activated by estrogen receptor mutants, we then validated their activity in ESR1-WT expressing cells stimulated with estrogen, as well as in the other ligand-binding domain mutants (ESR1-Y537N and ESR1-D538G) (FIG. 4-5). Our results demonstrate that these pathways are stimulated by ESR1-WT only in the presence of estrogen, but are constitutively active in mutant ESR1 expressing lines in the absence of estrogen stimulation.

[0062] Having demonstrated that these mutants can confer robust ESR1 canonical signaling that mimics estrogen stimulation, we next wanted to see if these receptors could compensate for loss of estrogen stimulation in ER+ breast cancer. To test this, we constructed a series of MCF-7 cells with expression of wild-type or mutated ESR1 and implanted these cells into male mice (low estrogen levels) without any exogenous estrogen stimulation. Asa control, we implanted both MCF-7 control cells (high ESR1) and MCF-7-ESR1-Y537N cells into female mice supplemented with exogenous estrogen. As predicted exogenous estrogen supplementation allowed for 100% tumor growth, while estrogen starvation resulted in strongly reduced tumor growth of control cells in 13% of mice and no control tumor growth in 87% of mice. This was in direct contrast to ESR1 mutant expressing cells which formed tumors in 80-100% of mice without exogenous estrogen supplementation (FIG. 6). However, ESR1-Y537N tumor growth in low estrogen conditions was somewhat slower non-supplemented mice, thus potentially demonstrating an effect from ESR1-WT present in MCF-7 cells. Collectively, these results demonstrate that these identified ESR1 mutants can compensate for suppression of estrogen in ER+ breast cancer in vivo, suggesting their efficacy as anti-estrogen therapies resistance mechanisms.

[0063] To determine if we could successfully immunologically target these mutated versions of ESR1 in a breast cancer setting, we first needed to establish a mouse model system of ER+ breast cancer. This model system would ideally have some form of transformation driven estrogen dependence whereby estrogen signaling was driven by these mutant versions of ESR1. To establish this model system, we first transduced MM3MG mammary breast epithelial cells with lentiviral vectors to stably express the various forms of ESR1. This mammary epithelial line was only weakly transformed (data not shown), expressed low endogenous levels of ESR1, and from a BALB/c background which would allow for immune studies to be performed in syngeneic BALB/c mice. After establishing these cell lines for each class of ESR1 mutant with a wild-type ESR1 control, we first confirmed that ERE signaling occurred in control lines after estrogen stimulation and in an ESR1 mutant line without estrogen stimulation (FIG. 7). To determine if these mutants had oncogenic capacity in these cells, we ascertained proliferation, anchorage-independent growth in soft agar, as well tumor formation and growth in vivo. Using MTT-based assays, we found that these mutants had no effect on proliferation (FIG. 8), although we did observe a significant increase in anchorage-independent growth when ESR1-Y537N was expressed in soft agar assays (FIG. 9). Finally, we injected ESR1-WT, ESR1-K303R, and ESR1-Y537N to determine the effect of ESR1-WT or mutated ESR1 on tumor growth in vivo. Notably, we found that both mutant ESR1 expressing lines grew more rapidly in these mice, demonstrating an oncogenic capacity of mutant estrogen signaling in these cells. Collectively, these results demonstrate that these mutants can confer estrogen-independent canonical signaling through several different pathways and that these mutant ESR1 can enhance murine mammary cell tumor growth in vivo.

[0064] Recent clinical studies have indicated that ESR1 mutations can be responsible for tamoxifen resistance in patients, which is supported by our findings using ESR1 mutant expressing cell lines. We hypothesized that immunologic targeting of these genes may prevent the development of resistance and potentially be a therapy for ER+ breast cancer. As such, we wished to explore the capacity of a vaccine targeting these oncogenic mutant forms of ESR1 to elicit anti-tumor responses. As a preliminary step, we took advantage of our adenoviral vector platform, which we had previously demonstrated to be capable of eliciting strong anti-tumor immunity against multiple Tumor Associated Antigens, including HER2. Using this platform, we constructed adenoviral vectors encoding wild-type and mutant forms of ESR1. After constructing and purifying these vectors, we ascertained their ability to elicit ESR-specific immunity in BALB/c mice. Using a ESR-specific ELISPOT assay, we determined that vaccination with ESR1 or mutant ESR1 genes strongly elicited significant T-cell mediated immunity to ESR1-specific epitopes (FIG. 11), while a ESR1-specific ELISA assay demonstrated significant ESR1 specific antibody responses (FIG. 12).

[0065] Having thus demonstrated these vaccines were capable of eliciting B-cell and T-cell ESR1-specific immunity, we next sought to determine if vaccination against a constitutively active oncogenic ESR1 mutant could significantly retard tumor growth. Having developed ESR1-mut transformed breast cancer lines capable of growing in immunocompetent transgenic animals, we next implanted these cells or ESR1-WT expressing counterparts into animals and tested if anti-ESR1 responses elicited by a preventative vaccination (2 weeks prior to injection) could retard ESR1-mediated growth. Our results demonstrated that an Ad-ESR1-Y537N vaccine formulation could significantly suppress ESR1-Y537N mediated tumor growth, but surprisingly did not affect MM3MG-ESR1-WT tumor growth (FIG. 13).

[0066] Additionally, we found that after a control Ad-GFP vaccination, MM3MG-ESR1-Y537N cells grew much more rapidly than MM3MG-ESR1-WT cells, thus demonstrating that an active form of ESR1 also enhanced growth in an immunocompetent setting. This was confirmed by ELISPOT assays that indicated Ad-ESR1-Y537N could elicit ESR1-specific T-cell responses in vaccinated tumor-bearing animals (FIG. 14). These results suggest that therapies targeting ESR1 mutants can have a significant immunologic impact on tumor growth in cancers driven by specific oncogenic mutations. As such, we expect that the use of checkpoint inhibitors in combination with our vaccine will allow for significant and sustained immune responses to critical oncogenic drivers and may prevent the development of resistance mediated by ESR1 mutation.

[0067] 1. Hartman, Z. C. et al. An adenovira vaccine encoding full-length inactivated human Her2 exhibits potent immunogenicty and enhanced therapeutic efficacy without oncogenicity. Clin. Cancer Res. 16, 1466-1477 (2010).

[0068] 2. Hartman, Z. C. et al. Growth of triple-negative breast cancer cells relies upon coordinate autocrine expression of the proinflammatory cytokines IL-6 and IL-8. Cancer Res 73, 3470-80 (2013).

[0069] 3. Hartman, Z. C. et al. HER2 overexpression elicits a proinflammatory IL-6 autocrine signaling loop that is critical for tumorigenesis. Cancer Re. 71, 4380-4391 (2011).

[0070] 4. Kershaw, M. H. et al. Gene-engineered T cells as a superior adjuvant therapy for metastatic cancer 1. J. Immunl. 173, 2143-2150 (2004).

[0071] 5. Hartman, Z. C. et al. Ligand-independent toll-like receptor signals generated by ectopic overexpression of MyD88 generate local and systemic antitumor immunity. Cancer Res. 70, 7209-7220 (2010).

[0072] 6. Early Breast Cancer Trialists' Collaborative, G. et al. Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet 378, 771-84 (2011).

[0073] 7. Baum, M. et al. Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early breast cancer: first results of the ATAC randomised trial. Lancet 359, 2131-9 (2002).

[0074] 8. Nabholtz, J. M. et al. Anastrozole is superior to tamoxifen as first-line therapy for advanced breast cancer in postmenopausal women: results of a North American multicenter randomized trial. Arimidex Study Group. J Clin Oncol 18, 3758-67 (2000).

[0075] 9. Ignatiadis, M. & Sotiriou, C. Luminal breast cancer: from biology to treatment. Nat Rev Clin Oncol 10, 494-506 (2013).

[0076] 10. Yerushalmi, R. et al. Tumor markers in metastatic breast cancer subtypes: frequency of elevation and correlation with outcome. Ann Oncol 23, 338-45 (2012).

[0077] 11. Yerushalmi, R. et al. Patterns of relapse in breast cancer: changes over time. Breast Cancer Res Treat 120, 753-9 (2010).

[0078] 12. Kennecke, H. et al. Metastatic behavior of breast cancer subtypes. J Clin Oncol 28, 3271-7(2010).

Sequence CWU 1

1

251595PRTHomo sapien 1Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His1 5 10 15Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr65 70 75 80Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105 110Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115 120 125Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly145 150 155 160Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170 175Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys225 230 235 240Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280 285Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn 290 295 300Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu305 310 315 320Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345 350Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355 360 365Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly385 390 395 400Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp465 470 475 480Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515 520 525Lys Cys Lys Asn Val Val Pro Leu Asn Asp Leu Leu Leu Glu Met Leu 530 535 540Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val545 550 555 560Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580 585 590Ala Thr Val 5952595PRTHomo sapien 2Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His1 5 10 15Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr65 70 75 80Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105 110Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115 120 125Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly145 150 155 160Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170 175Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys225 230 235 240Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280 285Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn 290 295 300Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu305 310 315 320Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345 350Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355 360 365Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly385 390 395 400Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp465 470 475 480Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515 520 525Lys Cys Lys Asn Val Val Pro Leu Ser Asp Leu Leu Leu Glu Met Leu 530 535 540Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val545 550 555 560Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580 585 590Ala Thr Val 5953595PRTHomo sapien 3Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His1 5 10 15Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr65 70 75 80Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105 110Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115 120 125Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly145 150 155 160Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170 175Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys225 230 235 240Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280 285Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn 290 295 300Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu305 310 315 320Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345 350Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355 360 365Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly385 390 395 400Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp465 470 475 480Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515 520 525Lys Cys Lys Asn Val Val Pro Leu Tyr Gly Leu Leu Leu Glu Met Leu 530 535 540Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val545 550 555 560Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580 585 590Ala Thr Val 59541788DNAHomo sapien 4atgaccatga ccctccacac caaagcatcc gggatggccc tactgcatca gatccaaggg 60aacgagctgg agcccctgaa ccgtccgcag ctcaagatcc ccctggagcg gcccctgggc 120gaggtgtacc tggacagcag caagcccgcc gtgtacaact accccgaggg cgccgcctac 180gagttcaacg ccgcggccgc cgccaacgcg caggtctacg gtcagaccgg cctcccctac 240ggccccgggt ctgaggctgc ggcgttcggc tccaacggcc tggggggttt ccccccactc 300aacagcgtgt ctccgagccc gctgatgcta ctgcacccgc cgccgcagct gtcgcctttc 360ctgcagcccc acggccagca ggtgccctac tacctggaga acgagcccag cggctacacg 420gtgcgcgagg ccggcccgcc ggcattctac aggccaaatt cagataatcg acgccagggt 480ggcagagaaa gattggccag taccaatgac aagggaagta tggctatgga atctgccaag 540gagactcgct actgtgcagt gtgcaatgac tatgcttcag gctaccatta tggagtctgg 600tcctgtgagg gctgcaaggc cttcttcaag agaagtattc aaggacataa cgactatatg 660tgtccagcca ccaaccagtg caccattgat aaaaacagga ggaagagctg ccaggcctgc 720cggctccgca aatgctacga agtgggaatg atgaaaggtg ggatacgaaa agaccgaaga 780ggagggagaa tgttgaaaca caagcgccag agagatgatg gggagggcag gggtgaagtg 840gggtctgctg gagacatgag agctgccaac ctttggccaa gcccgctcat gatcaaacgc 900tctaagagga acagcctggc cttgtccctg acggccgacc agatggtcag tgccttgttg 960gatgctgagc ccccgatact ctattccgag tatgatccta ccagaccctt cagtgaagct 1020tcgatgatgg gcttactgac caacctggca gacagggagc tggttcacat gatcaactgg 1080gcgaagaggg tgccaggctt tgtggatttg accctccatg atcaggtcca ccttctagaa 1140tgtgcctggc tagagatcct gatgattggt ctcgtctggc gctccatgga gcacccaggg 1200aagctactgt ttgctcctaa cttgctcttg gacaggaacc agggaaaatg tgtagagggc 1260atggtggaga tcttcgacat gctgctggct acatcatctc ggttccgcat gatgaatctg 1320cagggagagg agtttgtgtg cctcaaatct attattttgc ttaattctgg agtgtacaca 1380tttctgtcca gcaccctgaa gtctctggaa gagaaggacc atatccaccg agtcctggac 1440aagatcacag acactttgat ccacctgatg gccaaggcag gcctgaccct gcagcagcag 1500caccagcggc tggcccagct cctcctcatc ctctcccaca tcaggcacat gagtaacaaa 1560ggcatggagc atctgtacag catgaagtgc aagaacgtgg tgcccctcta tgacctgctg 1620ctggagatgc tggacgccca ccgcctacat gcgcccacta gccgtggagg ggcatccgtg 1680gaggagacgg accaaagcca cttggccact gcgggctcta cttcatcgca ttccttgcaa 1740aagtattaca tcacggggga ggcagagggt ttccctgcca cagtctag 17885595PRTHomo sapien 5Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His1 5 10 15Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr65 70 75 80Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105 110Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115 120 125Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly145 150 155 160Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170 175Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys225 230 235 240Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280 285Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Arg Asn 290 295 300Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu305 310 315 320Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345 350Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355 360 365Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 370 375

380Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly385 390 395 400Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp465 470 475 480Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515 520 525Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu 530 535 540Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val545 550 555 560Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580 585 590Ala Thr Val 59561239PRTHomo sapien 6Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu1 5 10 15Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys 20 25 30Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His 35 40 45Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val65 70 75 80Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln145 150 155 160Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys225 230 235 240Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260 265 270Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg 275 280 285Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln305 310 315 320Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe385 390 395 400Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val465 470 475 480Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485 490 495Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505 510Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys 515 520 525Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535 540Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys545 550 555 560Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620Pro Cys Pro Ile Asn Cys Thr His Ser Pro Leu Thr Ser Ile Val Ser625 630 635 640Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly 645 650 655Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg 660 665 670Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 675 680 685Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu 690 695 700Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys705 710 715 720Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile 725 730 735Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 740 745 750Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg 755 760 765Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu 770 775 780Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg785 790 795 800Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly 805 810 815Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala 820 825 830Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe 835 840 845Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp 850 855 860Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg865 870 875 880Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val 885 890 895Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 900 905 910Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 915 920 925Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met 930 935 940Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe945 950 955 960Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu 965 970 975Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu 980 985 990Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu 995 1000 1005Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala 1010 1015 1020Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg Ser 1025 1030 1035Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu 1040 1045 1050Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp 1055 1060 1065Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu Gln 1070 1075 1080Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser Glu 1085 1090 1095Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val Ala 1100 1105 1110Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro Asp 1115 1120 1125Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro Ala 1130 1135 1140Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Ala Lys Thr Leu Ser 1145 1150 1155Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly Gly 1160 1165 1170Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala Ala 1175 1180 1185Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp Asn 1190 1195 1200Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro Pro 1205 1210 1215Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr Leu 1220 1225 1230Gly Leu Asp Val Pro Val 12357111PRTHomo sapien 7Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu1 5 10 15Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys 20 25 30Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His 35 40 45Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val65 70 75 80Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn 100 105 1108668PRTHomo sapien 8Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu1 5 10 15Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys 20 25 30Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His 35 40 45Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val65 70 75 80Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln145 150 155 160Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys225 230 235 240Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260 265 270Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg 275 280 285Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln305 310 315 320Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe385 390 395 400Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val465 470 475 480Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485 490 495Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505 510Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys 515 520 525Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535 540Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys545 550 555 560Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620Pro Cys Pro Ile Asn Cys Thr His Ser Pro Leu Thr Ser Ile Val Ser625 630 635 640Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly 645 650 655Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys 660 6659331PRTHomo sapien 9Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu1 5 10 15Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 20 25 30Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 35 40 45Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 50 55 60Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile65 70 75 80Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 85 90 95Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 100 105 110Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 115 120 125His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 130 135 140Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr145 150 155 160Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 165 170 175Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 180 185 190Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 195 200 205Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 210 215 220Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser

Gly Pro Gln Asp225 230 235 240Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 245 250 255Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 260 265 270Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 275 280 285Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 290 295 300Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys305 310 315 320Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Phe 325 330101342PRTHomo sapien 10Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu1 5 10 15Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 20 25 30Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 35 40 45Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 50 55 60Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile65 70 75 80Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 85 90 95Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 100 105 110Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 115 120 125His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 130 135 140Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr145 150 155 160Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 165 170 175Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 180 185 190Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 195 200 205Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 210 215 220Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp225 230 235 240Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 245 250 255Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 260 265 270Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 275 280 285Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 290 295 300Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys305 310 315 320Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 325 330 335Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 340 345 350Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 355 360 365Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 370 375 380Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln385 390 395 400Ser Trp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr 405 410 415Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile 420 425 430Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu 435 440 445Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr 450 455 460His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu465 470 475 480Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu 485 490 495Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro 500 505 510Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val 515 520 525Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala 530 535 540His Glu Ala Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Gly545 550 555 560Gly Thr Ala Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys 565 570 575Ala His Phe Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly 580 585 590Val Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn 595 600 605Glu Cys Arg Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro 610 615 620Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr625 630 635 640His Leu Thr Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile Phe 645 650 655Met Met Leu Gly Gly Thr Phe Leu Tyr Trp Arg Gly Arg Arg Ile Gln 660 665 670Asn Lys Arg Ala Met Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu 675 680 685Pro Leu Asp Pro Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe 690 695 700Lys Glu Thr Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe705 710 715 720Gly Thr Val His Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys 725 730 735Ile Pro Val Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser 740 745 750Phe Gln Ala Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His 755 760 765Ala His Ile Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln 770 775 780Leu Val Thr Gln Tyr Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg785 790 795 800Gln His Arg Gly Ala Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val 805 810 815Gln Ile Ala Lys Gly Met Tyr Tyr Leu Glu Glu His Gly Met Val His 820 825 830Arg Asn Leu Ala Ala Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val 835 840 845Gln Val Ala Asp Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys 850 855 860Gln Leu Leu Tyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu865 870 875 880Glu Ser Ile His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser 885 890 895Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr 900 905 910Ala Gly Leu Arg Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu 915 920 925Arg Leu Ala Gln Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met 930 935 940Val Lys Cys Trp Met Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu945 950 955 960Leu Ala Asn Glu Phe Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu 965 970 975Val Ile Lys Arg Glu Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro 980 985 990His Gly Leu Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu 995 1000 1005Leu Asp Leu Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu Ala 1010 1015 1020Thr Thr Thr Leu Gly Ser Ala Leu Ser Leu Pro Val Gly Thr Leu 1025 1030 1035Asn Arg Pro Arg Gly Ser Gln Ser Leu Leu Ser Pro Ser Ser Gly 1040 1045 1050Tyr Met Pro Met Asn Gln Gly Asn Leu Gly Gly Ser Cys Gln Glu 1055 1060 1065Ser Ala Val Ser Gly Ser Ser Glu Arg Cys Pro Arg Pro Val Ser 1070 1075 1080Leu His Pro Met Pro Arg Gly Cys Leu Ala Ser Glu Ser Ser Glu 1085 1090 1095Gly His Val Thr Gly Ser Glu Ala Glu Leu Gln Glu Lys Val Ser 1100 1105 1110Met Cys Arg Ser Arg Ser Arg Ser Arg Ser Pro Arg Pro Arg Gly 1115 1120 1125Asp Ser Ala Tyr His Ser Gln Arg His Ser Leu Leu Thr Pro Val 1130 1135 1140Thr Pro Leu Ser Pro Pro Gly Leu Glu Glu Glu Asp Val Asn Gly 1145 1150 1155Tyr Val Met Pro Asp Thr His Leu Lys Gly Thr Pro Ser Ser Arg 1160 1165 1170Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Ser Val Leu Gly Thr 1175 1180 1185Glu Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg Arg 1190 1195 1200Arg Arg His Ser Pro Pro His Pro Pro Arg Pro Ser Ser Leu Glu 1205 1210 1215Glu Leu Gly Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser Ala 1220 1225 1230Ser Leu Gly Ser Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile 1235 1240 1245Met Pro Thr Ala Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr Met 1250 1255 1260Asn Arg Gln Arg Asp Gly Gly Gly Pro Gly Gly Asp Tyr Ala Ala 1265 1270 1275Met Gly Ala Cys Pro Ala Ser Glu Gln Gly Tyr Glu Glu Met Arg 1280 1285 1290Ala Phe Gln Gly Pro Gly His Gln Ala Pro His Val His Tyr Ala 1295 1300 1305Arg Leu Lys Thr Leu Arg Ser Leu Glu Ala Thr Asp Ser Ala Phe 1310 1315 1320Asp Asn Pro Asp Tyr Trp His Ser Arg Leu Phe Pro Lys Ala Asn 1325 1330 1335Ala Gln Arg Thr 1340119PRTHomo sapien 11Ile Gln Gly Asn Glu Leu Glu Pro Leu1 5129PRTHomo sapien 12Phe Met Val Leu Gln Val Ile Lys Phe1 5138PRTHomo sapien 13Leu Glu Met Leu Glu Ala Lys Val1 5148PRTHomo sapien 14Glu Val Phe Leu Pro Gln Arg Ala1 5159PRTHomo sapien 15Ile Phe Leu Asn Thr Glu Val Ser Leu1 5169PRTHomo sapien 16Leu Thr Ala Glu Glu Thr Asp Lys Ile1 51710PRTHomo sapien 17Leu Thr Ser Ser Ser Ile Asp Pro Gly Leu1 5 10189PRTHomo sapien 18Met Leu Lys His Lys Arg Pro Leu Ala1 51910PRTHomo sapien 19Thr Ile Val Ser Leu Asp Ala Ala Arg Arg1 5 10209PRTHomo sapien 20Lys Gly Asp Glu Glu Lys Glu Asn Asn1 5219PRTHomo sapien 21Leu Cys Val Lys Ala Met Ile Leu Leu1 5229PRTHomo sapien 22Met Asn Gln Lys Leu Ser Pro Phe Met1 5238PRTHomo sapien 23Arg Tyr Lys Lys Leu Lys Val Glu1 52410PRTHomo sapien 24Ser Lys Ala Lys Ser Leu Thr Asp Pro Ser1 5 10258PRTHomo sapien 25Trp Phe Gly Ile Lys Ala Pro Glu1 5

Claims

1. A vaccine comprising a polynucleotide encoding an ESR1 polypeptide, wherein the ESR1 polypeptide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 or a peptide fragment thereof at least eight amino acids long and comprising a mutation.

2. (canceled)

3. (canceled)

4. (canceled)

5. The vaccine of claim 1, further comprising a HER3 polypeptide or HER2 polypeptide or a mutant form of HER3 or HER2.

6. The vaccine of claim 1, further comprising a vaccine vector comprising the ESR1 polypeptide.

7. (canceled)

8. The vaccine of claim 6, wherein the vaccine vector is selected from adenovirus, adeno-associated virus (AAV), fowlpox, vaccinia virus, Venezuelen equine encephalitis virus.

9. (canceled)

10. The vaccine of claim 1, further comprising a checkpoint inhibitor immunomodulatory agent.

11. The vaccine of claim 10, wherein the checkpoint inhibitor immunomodulatory agent is a CTLA-4 or PD1 antagonistic antibody.

12. A method of treating a cancer or precancer or of reducing the likelihood of the cancer developing resistance to a cancer therapeutic or prevention agent comprising administering the vaccine of claim 1 to a subject having the cancer or precancer, wherein administration of the vaccine to the subject treats the cancer or precancer, reduces the likelihood of the cancer or precancer developing resistance to the cancer therapeutic or prevention agent or reverses resistance of the cancer or precancer to the cancer therapeutic or prevention agent.

13. (canceled)

14. The method of claim 12, wherein the vaccine is administered concurrently with, before or after administration of the cancer therapeutic or prevention agent.

15. The method of claim 14, wherein the cancer therapeutic or prevention agent is an agent targeting HER2, HER1, estrogen receptor, EGFR, or IGF1R.

16. The method of claim 12, wherein the vaccine is administered concurrently with, before or after administration of a checkpoint inhibitor immunomodulatory agent.

17. The method of claim 16, wherein the checkpoint inhibitor immunomodulatory agent is a CTLA-4 or PD1 antagonistic antibody.

18. The method of claim 12, wherein the cancer or precancer is selected from a breast, prostate, lung, ovarian, colon, rectal, pancreas, bladder, head and neck or liver cancer or precancer.

19. The method of claim 12, wherein the subject develops an immune response to ESR.

20. The method of claim 19, wherein the immune response comprises an antibody response or a T cell mediated response.

21. The method of claim 19, wherein the immune response includes at least one of antibody-dependent cellular cytotoxicity, polyclonal antibody response, complement dependent cellular cytotoxicity, cellular cytotoxicity, disruption of ligand binding, disruption of dimerization, mimicking ligand binding causing internalization of ESR1, or degradation of ESR.

22. The method of claim 19, wherein the immune response comprises an antibody response directed to at least a portion of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.

23. The method of claim 19, wherein the immune response is specific for a T cell epitope or a B cell epitope flanking or encompassing the mutation at position 537 of SEQ ID NO: 1, at position 538 of SEQ ID NO:3 or at position 303 of SEQ ID NO: 5.

24. The method of claim 12, wherein administration of the vaccine results in a reduction of ESR1 expression on cancer or precancer cells after administration of the vaccine as compared to the level of ESR1 on the cells prior to vaccination.

25. The method of claim 12, wherein administration results in decreased tumor growth rate or decreased tumor size after administration as compared to prior to administration.

26. The method of claim 12, wherein the cancer therapeutic or prevention agent is selected from trastuzumab, lapatinib, cetuximab, pertuzumab and erlotanib.