CHIMERIC ANTIGEN RECEPTOR DENDRITIC CELL (CAR-DC) FOR TREATMENT OF CANCER

Abstract

The current invention provides monocytic cells transfected with chimeric antigen receptor (CAR) to selectively home to tumors and upon homing differentiate into dendritic cells capable of activating immunity which is inhibitory to said tumor. In one embodiment of the invention, monocytic cells are transfected with a construct encoding an antigen binding domain, a transcellular or structural domain, and an intracellular signaling domain. In one specific aspect of the invention, the antigen binding domain interacts with sufficient affinity to a tumor antigen, capable of triggering said intracellular domain to induce an activation signal to induce monocyte differentiation into DC.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 62/118,027 filed on Feb. 19, 2015, the contents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure generally relates to the fields of genetics, immunology and medicine. The invention pertains to the field of immunotherapy, more specifically the invention pertains to the utilization of monocytes that have been manipulated to home to tumor cells and upon binding to tumor antigens differentiating into monocytes with cytotoxic properties to tumors, or dendritic cells.

BACKGROUND OF THE INVENTION

[0003] The immune system possesses the power to cure cancers based on published reports of immunologically mediated spontaneous regressions, which have been document in colon, lung, melanoma, liver, breast. Intriguingly, spontaneous regression clinically, as well as in an animal model of spontaneous regression, seems to be associated primarily with stimulation of the innate immune system, comprising of macrophages, NK cells, NKT cells and neutrophils. Despite the original promising of immunotherapy, which will be mentioned, the field has focused on the adaptive immune response, specifically stimulation of T and B cells, and only recently has interest re-ignited in the innate immune system.

[0004] The use of the immune system to treat cancer is theoretically appealing due to the possibility of low toxicity, immunological memory, and ability to attack metastatic disease. Early studies suggested that vaccination to tumor antigens and tumors themselves may be possible. Specifically, Prehn back in 1957, obtained murine tumors and exposed them to irradiation to increase immunogenicity. When these tumors were implanted into animals they were rejected, Subsequent administration of the original tumors resulted in rejection of the tumors, thus suggesting that tumor specific antigens exist, which can stimulate immunity, especially subsequent to addition of a cellular stress such as irradiation. Twenty years later, using the same system it was demonstrated that cytotoxic T cells infiltrated the tumors that were implanted after rejection of the radiation induced tumors, thus demonstrating conclusively that rejection was immunologically mediated, despite the fact that the tumors were syngeneic. In humans, one of the original observations of immunological response to neoplasia was in patients with paraneoplastic disease in which immune response to breast cancer antigens results in a multiple sclerosis-like disease caused by cross reactive immunity to neural antigens that are found on the breast cancer. Specific identification of tumor antigens on a molecular basis led to the discovery that some of the antigens are either self-proteins aberrantly expressed, or mutations of self proteins.

[0005] Originally observations were made in patients bearing metastatic melanomas, and then subsequently in other tumors, that the tumors are infiltrated with various immunological components. These tumor infiltrating lymphocytes (TILs), contain populations of cells and individual clones that demonstrate tumor specificity, they lyse autologous tumor cells but not natural killer targets, allogeneic tumor cells, or autologous fibroblasts.

[0006] By isolating and expanding TILs in vitro, and then molecularly identifying what they are responding to, a variety of the well-known tumor agents have been discovered such as MAGE-1, and MAGE-3, GAGE-1, MART-1, Melan-A, gp100, gp75 (TRP-2), tyrosinase, NY-ESO-1, mutated p16, and beta catenin. It is interesting that in the case of some antigens, such as gp75, the peptide that elicits tumor rejection results from translation of an alternative open reading frame of the same gene. Thus, the gp75 gene encodes two completely different polypeptides, gp75 as an antigen recognized by immunoglobulin G antibodies in sera from a patient with cancer, and a 24-amino acid product as a tumor rejection antigen recognized by T cells. Peptides used for immunization generally are 8-9 amino acids which have been demonstrated to be displayed in association with class I MHC molecules for recognition by T cells, and tumor cells have been shown to express these naturally processed epitopes.

[0007] Despite the intellectual appeal of peptide based cancer vaccines, the response rate has been disappointingly low. According to a review by Steven Rosenberg's group at the NIH, the rate of objective response out of 440 patients treated at his institute was a dismal 2.6%.

[0008] The ability to make a universal yet versatile system to generate T cells that are capable of recognizing various types of cancers has important clinical implications for the use of T cell-based therapies, this concept was approach initially by Rosenberg's group in the ex vivo expansion of tumor infiltrating lymphocytes. One current strategy incorporates the use of genetic engineering to express a chimeric antigen receptor (CAR) on T cells. The extracellular domain of a typical CAR consists of the V.sub.H and V.sub.L domains-single-chain fragment variable (scFv)--from the antigen binding sites of a monoclonal antibody. The scFv is linked to a flexible transmembrane domain followed by a tyrosine-based activation motif such as that from CD3.xi.. The so-called second and third generation CARs include additional activation domains from co-stimulatory molecules such as CD28 and CD137 (41BB) which serve to enhance T cell survival and proliferation. CAR T cells offer the opportunity to seek out and destroy cancer cells by recognizing tumor-associated antigens (TAA) expressed on their surface. As such, the recognition of a tumor cells occurs via an MHC-independent mechanism.

[0009] Various preclinical and early-phase clinical trials highlight the efficacy of CAR T cells to treat cancer patients with solid tumors and hematopoietic malignancies. Despite of the promise that CAR T cells might have in treating cancer patients there are several limitations to the generalized clinical application of CAR T cells. First, since no single tumor antigen is universally expressed by all cancer types, scFv in CAR needs to be constructed for each tumor antigen to be targeted. Second, the financial cost and labor-intensive tasks associated with identifying and engineering scFvs against a variety of tumor antigens poses a major challenge. Third, tumor antigens targeted by CAR could be down-regulated or mutated in response to treatment resulting in tumor evasion. Since current CAR T cells recognize only one target antigen, such changes in tumors negate the therapeutic effects. Therefore, the generation of CAR T cells capable of recognizing multiple tumor antigens is highly desired. Finally, CAR T cells react with target antigen weakly expressed on non-tumor cells, potentially causing severe adverse effects. To avoid such "on-target off-tumor" reaction, use of scFvs with higher specificity to tumor antigen is required. And although ongoing studies are focused on generating methods to shut off CAR T cells in vivo this system has yet to be developed and might pose additional inherent challenges.

[0010] The current patent seeks to apply chimeric antigen receptor technology to activation of monocytes, which naturally home into tumors, to differentiated intratumorally said monocytes into dendritic cells which are capable of antigen presentation, as well as direct killing of tumors.

DETAILED DESCRIPTION OF THE INVENTION

[0011] Chimeric antigen receptor (CAR) cellular therapeutics have revolutionized the treatment of B cell malignancies achieving stunning success rates. Unfortunately, solid tumors have yet to benefit from this treatment. Additionally, patients treated with CAR-T cells lack B cells for the rest of their lives, as well as having the possibility of tumor lysis syndrome. This is in part due to the permanence of the CAR-T cells in the patients after treatment. The current invention applies the use of CAR technology to monocytes with the purpose of inducing differentiation to dendritic cells (DC) subsequent to contact with tumor antigens. Given that monocytes have a fixed mitotic index, fears of permanent manipulation of the host are diminished.

[0012] "Treating a cancer", "inhibiting cancer", "reducing cancer growth" refers to inhibiting or preventing oncogenic activity of cancer cells. Oncogenic activity can comprise inhibiting migration, invasion, drug resistance, cell survival, anchorage-independent growth, non-responsiveness to cell death signals, angiogenesis, or combinations thereof of the cancer cells.

[0013] The terms "cancer", "cancer cell", "tumor", and "tumor cell" are used interchangeably herein and refer generally to a group of diseases characterized by uncontrolled, abnormal growth of cells (e.g., a neoplasia). In some forms of cancer, the cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body ("metastatic cancer").

[0014] "Ex vivo activated lymphocytes", "lymphocytes with enhanced antitumor activity" and "dendritic cell cytokine induced killers" are terms used interchangeably to refer to composition of cells that have been activated ex vivo and subsequently reintroduced within the context of the current invention. Although the word "lymphocyte" is used, this also includes heterogenous cells that have been expanded during the ex vivo culturing process including dendritic cells, NKT cells, gamma delta T cells, and various other innate and adaptive immune cells.

[0015] As used herein, "cancer" refers to all types of cancer or neoplasm or malignant tumors found in animals, including leukemias, carcinomas and sarcomas. Examples of cancers are cancer of the brain, melanoma, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus and Medulloblastoma.

[0016] The term "leukemia" is meant broadly progressive, malignant diseases of the hematopoietic organs/systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia diseases include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, and promyelocytic leukemi.

[0017] The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues, and/or resist physiological and non-physiological cell death signals and give rise to metastases. Exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrmcous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, and carcinoma scroti.

[0018] The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance. Sarcomas include, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilns' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma. Additional exemplary neoplasias include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer.

[0019] In some particular embodiments of the invention, the cancer treated is a melanoma. The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas include, for example, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.

[0020] The term "polypeptide" is used interchangeably with "peptide", "altered peptide ligand", and "flourocarbonated peptides."

[0021] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0022] The term "T cell" is also referred to as T lymphocyte, and means a cell derived from thymus among lymphocytes involved in an immune response. The T cell includes any of a CD8-positive T cell (cytotoxic T cell: CTL), a CD4-positive T cell (helper T cell), a suppressor T cell, a regulatory T cell such as a controlling T cell, an effector cell, a naive T cell, a memory T cell, an .alpha..beta.T cell expressing TCR .alpha. and .beta. chains, and a .gamma..delta. T cell expressing TCR .gamma. and .delta. chains. The T cell includes a precursor cell of a T cell in which differentiation into a T cell is directed.

[0023] Examples of "cell populations containing T cells" include, in addition to body fluids such as blood (peripheral blood, umbilical blood etc.) and bone marrow fluids, cell populations containing peripheral blood mononuclear cells (PBMC), hematopoietic cells, hematopoietic stem cells, umbilical blood mononuclear cells etc., which have been collected, isolated, purified or induced from the body fluids. Further, a variety of cell populations containing T cells and derived from hematopoietic cells can be used in the present invention. These cells may have been activated by cytokine such as IL-2 in vivo or ex vivo. As these cells, any of cells collected from a living body, or cells obtained via ex vivo culture, for example, a T cell population obtained by the method of the present invention as it is, or obtained by freeze preservation, can be used.

[0024] The term "antibody" is meant to include both intact molecules as well as fragments thereof that include the antigen-binding site. Whole antibody structure is often given as H.sub.2L.sub.2 and refers to the fact that antibodies commonly comprise 2 light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions interacting with the target are referred to as "variable" or "V" regions and are characterized by differences in amino acid sequence from antibodies of different antigenic specificity. The variable regions of either H or L chains contains the amino acid sequences capable of specifically binding to antigenic targets. Within these sequences are smaller sequences dubbed "hypervariable" because of their extreme variability between antibodies of differing specificity. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure. The CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have 3 CDR regions, each non-contiguous with the others (termed L1, L2, L3, H1, H2, H3) for the respective light (L) and heavy (H) chains. The antibodies disclosed according to the invention may also be wholly synthetic, wherein the polypeptide chains of the antibodies are synthesized and, possibly, optimized for binding to the polypeptides disclosed herein as being receptors. Such antibodies may be chimeric or humanized antibodies and may be fully tetrameric in structure, or may be dimeric and comprise only a single heavy and a single light chain.

[0025] The term "effective amount" or "therapeutically effective amount" means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic and/or physiologic effect, especially enhancing T cell response to a selected antigen. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being administered.

[0026] The terms "individual", "host", "subject", and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, primates, for example, human beings, as well as rodents, such as mice and rats, and other laboratory animals.

[0027] As used herein, the term "treatment regimen" refers to a treatment of a disease or a method for achieving a desired physiological change, such as increased or decreased response of the immune system to an antigen or immunogen, such as an increase or decrease in the number or activity of one or more cells, or cell types, that are involved in such response, wherein said treatment or method comprises administering to an animal, such as a mammal, especially a human being, a sufficient amount of two or more chemical agents or components of said regimen to effectively treat a disease or to produce said physiological change, wherein said chemical agents or components are administered together, such as part of the same composition, or administered separately and independently at the same time or at different times (i.e., administration of each agent or component is separated by a finite period of time from one or more of the agents or components) and where administration of said one or more agents or components achieves a result greater than that of any of said agents or components when administered alone or in isolation.

[0028] The term "anergy" and "unresponsiveness" includes unresponsiveness to an immune cell to stimulation, for example, stimulation by an activation receptor or cytokine. The anergy may occur due to, for example, exposure to an immune suppressor or exposure to an antigen in a high dose. Such anergy is generally antigen-specific, and continues even after completion of exposure to a tolerized antigen. For example, the anergy in a T cell and/or NK cell is characterized by failure of production of cytokine, for example, interleukin (IL)-2. The T cell anergy and/or NK cell anergy occurs in part when a first signal (signal via TCR or CD-3) is received in the absence of a second signal (costimulatory signal) upon exposure of a T cell and/or NK cell to an antigen.

[0029] The term "enhanced function of a T cell", "enhanced cytotoxicity" and "augmented activity" means that the effector function of the T cell and/or NK cell is improved. The enhanced function of the T cell and/or NK cell, which does not limit the present invention, includes an improvement in the proliferation rate of the T cell and/or NK cell, an increase in the production amount of cytokine, or an improvement in cytotoxity. Further, the enhanced function of the T cell and/or NK cell includes cancellation and suppression of tolerance of the T cell and/or NK cell in the suppressed state such as the anergy (unresponsive) state, or the rest state, that is, transfer of the T cell and/or NK cell from the suppressed state into the state where the T cell and/or NK cell responds to stimulation from the outside.

[0030] The term "expression" means generation of mRNA by transcription from nucleic acids such as genes, polynucleotides, and oligonucleotides, or generation of a protein or a polypeptide by transcription from mRNA. Expression may be detected by means including RT-PCR, Northern Blot, or in situ hybridization.

[0031] "Suppression of expression" refers to a decrease of a transcription product or a translation product in a significant amount as compared with the case of no suppression. The suppression of expression herein shows, for example, a decrease of a transcription product or a translation product in an amount of 30% or more, preferably 50% or more, more preferably 70% or more, and further preferably 90% or more.

[0032] In one embodiment of the invention the CAR-DC are antigen-loaded and co-cultured with T-lymphocytes to produce antigen-specific T-cells. As used herein, the term "antigen-specific T-cells" refers to T-cells that proliferate upon exposure to the antigen-loaded APCs of the present invention, as well as to develop the ability to attack cells having the specific antigen on their surfaces. Such T-cells, e.g., cytotoxic T-cells, lyse target cells by a number of methods, e.g., releasing toxic enzymes such as granzymes and perforin onto the surface of the target cells or by effecting the entrance of these lytic enzymes into the target cell interior. Generally, cytotoxic T-cells express CD8 on their cell surface. T-cells that express the CD4 antigen CD4, commonly known as "helper" T-cells, can also help promote specific cytotoxic activity and may also be activated by the antigen-loaded APCs of the present invention. In certain embodiments, the cancer cells, the APCs and even the T-cells can be derived from the same donor whose MNC yielded the DC, which can be the patient or an HLA-or obtained from the individual patient that is going to be treated. Alternatively, the cancer cells, the APCs and/or the T-cells can be allogeneic.

[0033] The invention provides means of inducing an anti-cancer response in a mammal, comprising the steps of initially "priming" the mammal by administering an agent that causes local accumulation of CAR-DC. Subsequently, a tumor antigen is administered in the local area where said agents causing accumulation of antigen presenting cells is administered. A time period is allowed to pass to allow for said antigen presenting cells to traffic to the lymph nodes. Subsequently a maturation signal, or a plurality of maturation signals are administered to enhance the ability of said antigen presenting cell to activate adaptive immunity. In some embodiments of the invention activators of adaptive immunity are concurrently given, as well as inhibitors of the tumor derived inhibitors are administered to derepress the immune system.

[0034] In one embodiment priming of the patient is achieved by administration of GM-CSF subcutaneously in the area in which antigen is to be injected. Various scenarios are known in the art for administration of GM-CSF prior to administration, or concurrently with administration of antigen. The practitioner of the invention is referred to the following publications for dosage regimens of GM-CSF and also of peptide antigens.

[0035] Subsequent to priming, the invention calls for administration of tumor antigen. Various tumor antigens may be utilized, in one preferred embodiment, lysed tumor cells from the same patient area utilized. Means for generation of lyzed tumor cells are well known in the art and described in the following references. One example method for generation of tumor lysate involves obtaining frozen autologous samples which are placed in hanks buffered saline solution (HBSS) and gentamycin 50 .mu.g/ml followed by homogenization by a glass homogenizer. After repeated freezing and thawing, particle-containing samples are selected and frozen in aliquots after radiation with 25 kGy. Quality assessment for sterility and endotoxin content is performed before freezing. Cell lysates are subsequently administered into the patient in a preferred manner subcutaneously at the local areas where DC priming was initiated. After 12-72 hours, the patient is subsequently administered with an agent capable of inducing maturation of DC. Agents useful for the practice of the invention, in a preferred embodiment include BCG and HMGB1 peptide. Other useful agents include: a) histone DNA; b) imiqimod; c) beta-glucan; d) hsp65; e) hsp90; f) HMGB-1; g) lipopolysaccharide; h) Pam3CSK4; i) Poly I: Poly C; j) Flagellin; k) MALP-2; I) Imidazoquinoline; m) Resiquimod; n) CpG oligonucleotides; o) zymosan; p) peptidoglycan; q) lipoteichoic acid; r) lipoprotein from gram-positive bacteria; s) lipoarabinomannan from mycobacteria; t) Polyadenylic-polyuridylic acid; u) monophosphoryl lipid A; v) single stranded RNA; w) double stranded RNA; x) 852A; y) rintatolimod; z) Gardiquimod; and aa) lipopolysaccharide peptides. The procedure is performed in a preferred embodiment with the administration of IDO silencing siRNA or shRNA containing the effector sequences a) UUAUAAUGACUGGAUGUUC; b) GUCUGGUGUAUGAAGGGUU; c) CUCCUAUUUUGGUUUAUGC and d) GCAGCGUCUUUCAGUGCUU. siRNA or shRNA may be administered through various modalities including biodegradable matrices, pressure gradients or viral transfect. In another embodiment, autologous dendritic cells are generated and IDO is silenced, prior to, concurrent with or subsequent to silencing, said dendritic cells are pulsed with tumor antigen and administered systemically.

[0036] In one embodiment of the invention mature DC are modified with CAR transfection prior to administration. Culture of dendritic cells is well known in the art, for example, U.S. Pat. No. 6,936,468, issued to Robbins, et al., for the use of tolerogenic dendritic cells for enhancing tolerogenicity in a host and methods for making the same. Although the current invention aims to reduce tolerogenesis, the essential means of dendritic cell generation are disclosed in the patent. U.S. Pat. No. 6,734,014, issued to Hwu, et al., for methods and compositions for transforming dendritic cells and activating T cells. Briefly, recombinant dendritic cells are made by transforming a stem cell and differentiating the stem cell into a dendritic cell. The resulting dendritic cell is said to be an antigen presenting cell which activates T cells against MHC class I-antigen targets. Antigens for use in dendritic cell loading are taught in, e.g., U.S. Pat. No. 6,602,709, issued to Albert, et al. This patent teaches methods for use of apoptotic cells to deliver antigen to dendritic cells for induction or tolerization of T cells. The methods and compositions are said to be useful for delivering antigens to dendritic cells that are useful for inducing antigen-specific cytotoxic T lymphocytes and T helper cells. The disclosure includes assays for evaluating the activity of cytotoxic T lymphocytes. The antigens targeted to dendritic cells are apoptotic cells that may also be modified to express non-native antigens for presentation to the dendritic cells. The dendritic cells are said to be primed by the apoptotic cells (and fragments thereof) capable of processing and presenting the processed antigen and inducing cytotoxic T lymphocyte activity or may also be used in vaccine therapies. U.S. Pat. No. 6,455,299, issued to Steinman, et al., teaches methods of use for viral vectors to deliver antigen to dendritic cells. Methods and compositions are said to be useful for delivering antigens to dendritic cells, which are then useful for inducing T antigen specific cytotoxic T lymphocytes. The disclosure provides assays for evaluating the activity of cytotoxic T lymphocytes. Antigens are provided to dendritic cells using a viral vector such as influenza virus that may be modified to express non-native antigens for presentation to the dendritic cells. The dendritic cells are infected with the vector and are said to be capable of presenting the antigen and inducing cytotoxic T lymphocyte activity or may also be used as vaccines.

[0037] Immune cells for use in the practice of the invention include DCs, the presence of which may be checked in the previously described method, are preferably selected from myeloid cells (such as monocytic cells and macrophages) expressing langerin, MHC (major histocompatibility complex) class II, CCR2 (chemokine (C--C motif) receptor 2), CX3CR1 and/or Gr1 molecules in mice; myeloid cells expressing CD14, CD16, HLA dR (human leukocyte antigen disease resistance) molecule, langerin, CCR2 and/or CX3CR1 in humans; dendritic cells expressing CD11c, MHC class II molecules, and/or CCR7 molecules; and IL-1.beta. producing dendritic cells. CD8 T cells, the presence of which may be checked in the previously described method, are preferably selected from CD3+, CD4+ and/or CD8+ T lymphocytes, FOXP3 (forkhead box P3) T lymphocytes, Granzyme B/TIA (Tcell-restricted intracellular antigen) T lymphocytes, and Tc1 cells (IFN-.gamma. producing CD8+ T lymphocytes). Immune cells expressing a protein that binds calreticulin, such immune cells may be selected from cells expressing at least one of the following proteins: LRP1 (Low density lipoprotein receptor-related protein 1, CD91), Ca.sup.++-binding proteins such as SCARF1 and SCARF2, MSR1 (Macrophage scavenger receptor 1), SRA, CD59 (protectin), CD207 (langerin), and THSD1 (thrombospondin). There are numerous means known in the art to identify cells expressing various antigens, these include immunochemistry, immunophenotyping, flow cytometry, Elispots assays, classical tetramer staining, and intracellular cytokine stainings.

[0038] Macrophages selectively phagocytose tumor cells, but this process is countered by protective molecules on tumor cells such as CD47, which binds macrophage signal-regulatory protein a to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. In one embodiment of the invention CAR-MSC are administered together with an agent that blocks CD47 activity. It has been demonstrated that activation of TLR signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance tumor phagocytosis. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Careticulin, previously shown to be a protein found on cancer cells that activated macrophage phagocytosis of tumors, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin. In one embodiment of the invention TLR agonists are administered that stimulate expression of calreticulin and/or enhance macrophage phagocytosis of tumors.

[0039] IL-27 induces macrophage ability to kill tumor cells in vitro and in vivo, as well as altering the tumor promoting M2/myeloid suppressor cells into tumoricidal cells. In one embodiment of the invention addition of IL-27 or compounds capable of activating the IL-27 receptor signaling are administered together with IL-27 to enhance tumor phagocytosis by macrophages.

[0040] Tumor-associated macrophages, deriving from monocytes or migrating into the tumor, are an important constituent of tumor microenvironments, which in many cases modulates tumor growth, tumor angiogenesis, immune suppression, metastasis and chemoresistance. Mechanisms of macrophage promotion of tumor growth include production of EGF, M-CSF, VEGF.

[0041] Macrophage infiltration of tumors is associated with poor prognosis in renal, melanoma, breast, pancreatic, lung, endometrial, bladder, prostate.

[0042] Tumor growth are inhibited when monocytes/macrophages are ablated. There is ample evidence that many anticancer modalities currently used in the clinic have unique and distinct properties that modulate the recruitment, polarization and tumorigenic activities of macrophages in the tumor microenvironments. By manipulating tumor-associated macrophages significant impact on the clinical efficacies of and resistance to these anticancer modalities. Accordingly, in one aspect of the invention, CAR-DC, CAR-monocytes, or CAR-macrophages are utilized to force the tumor microenvironment to stimulate tumor killing and inhibit macrophage or macrophage related cells from promoting tumor growth. Within the context of the invention, the use of drugs targeting tumor-associated macrophages, especially c-Fms kinase inhibitors and humanized antibodies targeting colony-stimulating factor-1 receptor, are envisioned.

[0043] Tumors mediate various effects to reprogram macrophages, these are usually mediated via IL-10 and other cytokines such as VEGF, TGF-beta, and M-CSF, which cause macrophages to lose tumor cytotoxicity and shift into tumor promoting, immune suppressive, angiogenic supporting cells. Related to tumor manipulated monocytes are myeloid derived suppressor cells, which are similar to myeloid progenitor cells, or the previously described "natural suppressor" cell.

[0044] Irradiated tissues induce a TLR-1 reprogramming of macrophages to promote tumor growth and angiogenesis. Macrophage promotion of tumor growth is seen in numerous situations, in one example, treating of tumor bearing animals with BRAF inhibitors results in upregulation of macrophage production of VEGF which accelerates tumor growth. Mechanistically, it is known that tumors produce factors such as GM-CSF which in part stimulate macrophages to produce CCL18, which promotes tumor metastasis. Additionally, the lactic acid microenvironment of the tumor has been shown to promote skewing of macrophages towards at tumor-promoting M2 type. It has been shown that lactic acid produced by tumour cells, as a by-product of aerobic or anaerobic glycolysis, possesses an essential role in inducing the expression of VEGF and the M2-like polarization of tumour-associated macrophages, specifically inducing expression of arginase 1 through a HIF-1alpha dependent pathway. Mechanistically, it is known that lactic acid in tumors is generated in a large part by lactate dehydrogenase-A (LDH-A), which converts pyruvate to lactate. siRNA silencing of LDH-A in Pan02 pancreatic cancer cells that are injected in C57BL/6 mice results in development of smaller tumors than mice injected with wild type, non-silenced Pan02 cells. Associated with the reduced tumor growth were observations of a decrease in the frequency of myeloid-derived suppressor cells (MDSCs) in the spleens of mice carrying LDH-A-silenced tumors. NK cells from LDH-A-depleted tumors had improved cytolytic function. Exogenous lactate administration was shown to increase the frequency of MDSCs generated from mouse bone marrow cells with GM-CSF and IL-6 in vitro. Furthermore lactate pretreatment of NK cells in vitro inhibited cytolytic function of both human and mouse NK cells. This reduction of NK cytotoxic activity was accompanied by lower expression of perforin and granzyme in NK cells. The expression of NKp46 was lower in lactate-treated NK cells. Accordingly, in one embodiment of the invention, depletion of glucose levels using a ketogenic diet to lower lactate production by glycolytic tumors is utilized to augment therapeutic effects of CAR-DC. Utilization of ketogenic diet has been previously described for immune modulation, and cancer therapy. Specific quantification of intratumoral lactate and its manipulation has been described and incorporated by reference. Potentiation of chemotherapeutic and radiotherapeutic effects by ketogenic diets have been reported and techniques are incorporated by reference for use with the current CAR-DC invention. Suppression of tumor growth and activity induced by ketogenic diet may be augmented by addition of hyperbaric oxygen, thus in one embodiment of the invention, the utilization of oxidative therapies, as disclosed in references incorporated, together with ketogenic diet is utilized to augment therapeutic efficacy of CAR-DC.

[0045] Not only has it been well known that monocytes and macrophages infiltrate tumors and appear to support tumor growth through growth factor production and secretion of angiogenic agents, but suggestions have been made that tumors themselves, as part of the epithelial mesenchymal transition may actually differentiate into monocytes in part associated with TGF-beta production. Specifically, a study reported performing gene-profiling analysis of mouse mammary EpRas tumor cells that had been allowed to adopt an epithelial to mesenchymal transition program after long-term treatment with TGF-.beta.1 for 2 weeks. While the treated cells acquired traits of mesenchymal cell differentiation and migration, gene analysis revealed another cluster of induced genes, which was specifically enriched in monocyte-derived macrophages, mast cells, and myeloid dendritic cells, but less in other types of immune cells. Further studies revealed that this monocyte/macrophage gene cluster was enriched in human breast cancer cell lines displaying an EMT or a Basal B profile, and in human breast tumors with EMT and undifferentiated (ER-/PR-) characteristics. The plasticity of tumor cells to potentially monocytic lineages should come as no surprise given that tumor cells have been shown to differentiate directly into pericytes, and endothelial cells/vascular channels.

[0046] Dopamine possesses antiangiogenic effects as well as myeloprotective effects, in one embodiment of the invention addition of dopamine to the CAR-DC treatment is disclosed.

[0047] Vinblastine is a widely used chemotherapeutic agent that has been demonstrated to induce dendritic cell maturation. In one embodiment of the invention CAR-DC are utilized together with vinblastine therapy to induce augmented anticancer activity. Oxiplatin and anthracyclines have been demonstrated to not only directly kill tumor cells but also stimulate T cell immunity against tumor cells. It was demonstrated that these agents induce a rapid and prominent invasion of interleukin (IL)-17-producing .gamma..delta. (V.gamma.4(+) and V.gamma.6(+)) T lymphocytes (.gamma..delta. T17 cells) that precedes the accumulation of CD8 CTLs within the tumor bed. In T cell receptor .delta.(-/-) or V.gamma.4/6(-/-) mice, the therapeutic efficacy of chemotherapy was reduced and furthermore no IL-17 was produced by tumor-infiltrating T cells, and CD8 CTLs did not invade the tumor after treatment. Although .gamma..delta. Th17 cells could produce both IL-17A and IL-22, the absence of a functional IL-17A-IL-17R pathway significantly reduced tumor-specific T cell responses elicited by tumor cell death, and the efficacy of chemotherapy in four independent transplantable tumor models. The adoptive transfer of .gamma..delta. T cells to naive mice restored the efficacy of chemotherapy in IL-17A(-/-) hosts. The anticancer effect of infused .gamma..delta. T cells was lost when they lacked either IL-1R1 or IL-17A.

[0048] Intratumoral injection of dendritic cells stimulates antitumor immunity in vivo in clinical situations, suggesting that modulating the antigen presenting cell in the tumor microenvironment will induce an antitumor response. Administration of radiotherapy to tumors to induce immunogenic cell death, followed by intratumoral administration of DC has been demonstrated to result in enhanced antigen presentation, accordingly, this technique may be modified to enhance effects of CAR-DC. The induction of immunity to tumors in the present invention is associated with the unique nature of: a) ongoing basal cell death within the tumor; and b) cell death induced by chemotherapy, radiotherapy, hyperthermia, or otherwise induced cell death. Cell death can be classified according to the morphological appearance of the lethal process (that may be apoptotic, necrotic, autophagic or associated with mitosis), enzymological criteria (with and without the involvement of nucleases or distinct classes of proteases, like caspases), functional aspects (programmed or accidental, physiological or pathological) or immunological characteristics (immunogenic or non-immunogenic). Cell death is defined as "immunogenic" or "immune stimulatory" if dying cells that express a specific antigen (for example a tumor associated antigen, phosphotidyl serine, or calreticulin), yet are uninfected (and hence lack pathogen-associated molecular patterns), and are injected subcutaneously into mice, in the absence of any adjuvant, cause a protective immune response against said specific antigen. Such a protective immune response precludes the growth of living transformed cells expressing the specific antigen injected into mice. When cancer cells succumb to an immunogenic cell death (or immunogenic apoptosis) modality, they stimulate the immune system, which then mounts a therapeutic anti-cancer immune response and contributes to the eradication of residual tumor cells. Conversely, when cancer cells succumb to a non-immunogenic death modality, they fail to elicit such a protective immune response. Regardless of the types of cell death that are ongoing, the tumor derived immune suppressive molecules contribute to general inhibition or inability of the tumor to be eliminated.

[0049] Within the practice of the invention, CAR-DC are administered concurrently, prior to, or subsequent to administration of an agent that induces immunogenic cell death in a patient. Methods of determining whether compounds induce immunogenic cell death are known in the art and include the following, which was described by Zitvogel et al. (a) treating the cells, the mammalian cells and inducing the cell death or apoptosis, typically of mammalian cancer cells capable of expressing calreticulin (CRT), by exposing said mammalian cells to a particular drug (the test drug), for example 18 hours; (b) inoculating (for example intradermally) the dying mammalian cells from step (a) in a particular area (for example a flank) of the mammal, typically a mouse, to induce an immune response in this area of the mammal; (c) inoculating (for example intradermally) the minimal tumorigenic dose of syngeneic live tumor cells in a distinct area (for example the opposite flank) from the same mammal, for example 7 days after step (b); and (d) comparing the size of the tumor in the inoculated mammal with a control mammal also exposed to the minimal tumorigenic dose of syngeneic live tumor cells of step (c) [for example a mouse devoid of T lymphocyte], the stabilization or regression of the tumor in the inoculated mammal being indicative of the drug immunogenicity. Other in vitro means are available for assessing the ability of various drugs or therapeutic approaches to induce immunogenic cell death. Specific characteristics to assess when screening for immunogenic cell death include: a) ability to induce dendritic cell maturation in vitro; b) ability to activate NK cells; and c) ability to induce activation of gamma delta T cells or NKT cells. Specific drugs known to induce immunogenic cell death include oxiplatine and anthracyclines, as well as radiotherapy, and hyperthermia. In the case of chemotherapies, certain chemotherapies that activate TLR4 through induction of HMGB1 have been observed to function suboptimally in patients that have a TLR4 polymorphism, thus suggesting actual contribution of TLR activation as a means of chemotherapy inhibition of cancer. Additionally, oncoviruses or oncolytic viruses are known to induce immunogenic cell death and may be useful for the practice of the invention.

[0050] The CAR-DC disclosed in the invention may be utilized in combination with conventional immune modulators including BCG, CpG DNA, interferon alpha, tumor bacterial therapy, checkpoint inhibitors, Treg depleting agents, and low dose cyclophosphamide.

[0051] In one embodiment of the invention CAR-DC cells are generated with specificity towards ROBO-4. Numerous means of generating CAR-T cells are known in the art, which are applied to CAR-DC. In one embodiment of the invention FMC63-28z CAR (Genebank Identifier HM852952.1), is used as the template for the CAR except the anti-CD19, single-chain variable fragment sequence is replaced with an ROBO-4 fragment. The construct is synthesized and inserted into a pLNCX retroviral vector. Retroviruses encoding the ROBO-4-specific CAR are generated using the retrovirus packaging kit, Ampho (Takara), following the manufacturer's protocol. For generation of CAR-DC cells donor blood is obtained and after centrifugation on Ficoll-Hypaque density gradients (Sigma-Aldrich), PBMCs are plated at 2.times.10(6) cells/mL in cell culture for 2 hours and the adherent cells are collected. The cells were then stimulated for 2 days on a tissue-culture-treated 24-well plate containing M-CSF at a concentration of 100 ng/ml For retrovirus transduction, a 24-well plate are coated with RetroNectin (Takara) at 4.degree. C. overnight, according to the manufacturer's protocol, and then blocked with 2% BSA at room temperature for 30 min. The plate was then loaded with retrovirus supernatants at 300 .mu.L/well and incubated at 37.degree. C. for 6 h. Next, 1.times.10(6) stimulated adherent cells in 1 mL of medium are added to 1 mL of retrovirus supernatants before being transferred to the pre-coated wells and cultured at 37.degree. C. for 2 d. The cells are then transferred to a tissue-culture-treated plate at 1.times.10 (6) cells/mL and cultured in the presence of 100 U/mL of recombinant human M-CSF, applying the T cell protocol but not utilizing IL-2 or antiCD3/antiCD28.

[0052] Other means of generating CARs are known in the art and incorporated by reference. For example, Groner's group genetically modified T lymphocytes and endowed them with the ability to specifically recognize cancer cells. Tumor cells overexpressing the ErbB-2 receptor served as a model. The target cell recognition specificity was conferred to T lymphocytes by transduction of a chimeric gene encoding the zeta-chain of the TCR and a single chain antibody (scFv(FRP5)) directed against the human ErbB-2 receptor. The chimeric scFv(FRP5)-zeta gene was introduced into primary mouse T lymphocytes via retroviral gene transfer. Naive T lymphocytes were activated and infected by cocultivation with a retrovirus-producing packaging cell line. The scFv(FRP5)-zeta fusion gene was expressed in >75% of the T cells. These T cells lysed ErbB-2-expressing target cells in vitro with high specificity. In a syngeneic mouse model, mice were treated with autologous, transduced T cells. The adoptively transferred scFv(FRP5)-zeta-expressing T cells caused total regression of ErbB-2-expressing tumors. The presence of the transduced T lymphocytes in the tumor tissue was monitored. No humoral response directed against the transduced T cells was observed. Abs directed against the ErbB-2 receptor were detected upon tumor lysis. Hombach et al. constructed an anti-CEA chimeric receptor whose extracellular moiety is composed of a humanized scFv derived from the anti-CEA mAb BW431/26 and the CH2/CH3 constant domains of human IgG. The intracellular moiety consists of the gamma-signaling chain of the human Fc epsilon RI receptor constituting a completely humanized chimeric receptor. After transfection, the humBW431/26 scFv-CH2CH3-gamma receptor is expressed as a homodimer on the surface of MD45 T cells. Co-incubation with CEA+ tumor cells specifically activates grafted MD45 T cells indicated by IL-2 secretion and cytolytic activity against CEA+ tumor cells. Notably, the efficacy of receptor-mediated activation is not affected by soluble CEA up to 25 micrograms/ml demonstrating the usefulness of this chimeric receptor for specific cellular activation by membrane-bound CEA even in the presence of high concentrations of CEA, as found in patients during progression of the disease (200). These methods are described to guide one of skill in the art to practicing the invention, which in one embodiment is the utilization of CAR T cell approaches towards targeting tumor endothelium as comparted to simply targeting the tumor itself.

[0053] Targeting of mucins associated with cancers has been performed with CAR T cells by grafting the antibody that binds to the mucin with CD3 zeta chain. For the purpose of the invention, this procedure is modified for CAR-DC. In an older publication chimeric immune receptor consisting of an extracellular antigen-binding domain derived from the CC49 humanized single-chain antibody, linked to the CD3zeta signaling domain of the T cell receptor, was generated (CC49-zeta). This receptor binds to TAG-72, a mucin antigen expressed by most human adenocarcinomas. CC49-zeta was expressed in CD4+ and CD8+ T cells and induced cytokine production on stimulation. Human T cells expressing CC49-zeta recognized and killed tumor cell lines and primary tumor cells expressing TAG-72. CC49-zeta T cells did not mediate bystander killing of TAG-72-negative cells. In addition, CC49-zeta T cells not only killed FasL-positive tumor cells in vitro and in vivo, but also survived in their presence, and were immunoprotective in intraperitoneal and subcutaneous murine tumor xenograft models with TAG-72-positive human tumor cells. Finally, receptor-positive T cells were still effective in killing TAG-72-positive targets in the presence of physiological levels of soluble TAG-72, and did not induce killing of TAG-72-negative cells under the same conditions.

[0054] For clinical practice of the invention several reports exist in the art that would guide the skilled artisan as to concentrations, cell numbers, and dosing protocols useful. While in the art CAR T cells have been utilized targeting surface tumor antigens, the main issue with this approach is the difficulty of T cells to enter tumors due to features specific to the tumor microenvironment. These include higher interstitial pressure inside the tumor compared to the surroundings, acidosis inside the tumor, and expression in the tumor of FasL which kills activated T cells. Accordingly the invention seeks to more effectively utilize CAR-DC cells by directly targeting them to tumor endothelium, which is in direct contact with blood and therefore not susceptible to intratumoral factors the limit efficacy of conventional T cell therapies. In other embodiments CAR-DC are targeting to tumor antigens.

[0055] In one embodiment of the invention, protocols similar to Kershaw et al. are utilized with the exception that tumor endothelial antigens are targeted as opposed to conventional tumor antigens. Such tumor endothelial antigens include CD93, TEM-1, VEGFR1, and survivin. Antibodies can be made for these proteins, methodologies for which are described in U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761, and 5,639,641. In one example that may be utilized as a template for clinical development, T cells with reactivity against the ovarian cancer-associated antigen alpha-folate receptor (FR) were generated by genetic modification of autologous T cells with a chimeric gene incorporating an anti-FR single-chain antibody linked to the signaling domain of the Fc receptor gamma chain. Patients were assigned to one of two cohorts in the study. Eight patients in cohort 1 received a dose escalation of T cells in combination with high-dose interleukin-2, and six patients in cohort 2 received dual-specific T cells (reactive with both FR and allogeneic cells) followed by immunization with allogeneic peripheral blood mononuclear cells. Five patients in cohort 1 experienced some grade 3 to 4 treatment-related toxicity that was probably due to interleukin-2 administration, which could be managed using standard measures. Patients in cohort 2 experienced relatively mild side effects with grade 1 to 2 symptoms. No reduction in tumor burden was seen in any patient. Tracking 111In-labeled adoptively transferred T cells in cohort 1 revealed a lack of specific localization of T cells to tumor except in one patient where some signal was detected in a peritoneal deposit. PCR analysis showed that gene-modified T cells were present in the circulation in large numbers for the first 2 days after transfer, but these quickly declined to be barely detectable 1 month later in most patients. Similar CAR-T clinical studies have been reported for neuroblastoma, B cell malignancies, melanoma, ovarian cancer, renal cancer, mesothelioma, and head and neck cancer.

[0056] In one embodiment of the invention, PBMCs are derived from leukapheresis and CD14 monocytes are collected by MACS. After 3 days of culture, M-CSF at 100 ng/ml plasmid encoding the chimeric CAR-DC recognizing tumor-endothelium specific antigen and subsequently selected for gene integration by culture in G418. In another embodiment the generation of dual-specific T cells is performed, stimulation of allogeneic monocytic cells is achieved by coculture of patient PBMCs with irradiated (5,000 cGy) allogeneic donor PBMCs from cryopreserved apheresis product (mixed lymphocyte reaction). The MHC haplotype of allogeneic donors is determined before use, and donors that differed in at least four MHC class I alleles from the patient are used. Culture medium consisted of AimV medium (Invitrogen, Carlsbad, Calif.) supplemented with 5% human AB.sup.- serum (Valley Biomedical, Winchester, Va.), penicillin (50 units/mL), streptomycin (50 mg/mL; Bio Whittaker, Walkersville, Md.), amphotericin B (Fungizone, 1.25 mg/mL; Biofluids, Rockville, Md.), L-glutamine (2 mmol/L; Mediatech, Herndon, Va.), and human recombinant IL-2 (Proleukin, 300 IU/mL; Chiron). Mixed lymphocyte reaction consisted of 2.times.10.sup.6 patient monocytes and 1.times.10.sup.7 allogeneic stimulator PBMCs in 2 mL AimV per well in 24-well plates. Between 24 and 48 wells are cultured per patient for 3 days, at which time transduction is done by aspirating 1.5 mL of medium and replacing with 2.0 mL retroviral supernatant containing 300 IU/ml IL-2, 10 mmol/L HEPES, and 8 .mu.g/mL polybrene (Sigma, St. Louis, Mo.) followed by covering with plastic wrap and centrifugation at 1,000.times.g for 1 hour at room temperature. After overnight culture at 37.degree. C./5% CO.sub.2, transduction is repeated on the following day, and then medium was replaced after another 24 hours. Cells are then resuspended at 1.times.10.sup.8/mL in fresh medium containing 0.5 mg/mL G418 (invitrogen) in 175-cm.sup.2 flasks for 5 days before resuspension in media lacking G418. `Cells are expanded to 2.times.10.sup.9 and then restimulated with allogeneic PBMCs from the same donor to enrich for T cells specific for the donor allogeneic haplotype. Restimulation is done by incubating patient T cells (1.times.10.sup.6/mL) and stimulator PBMCs (2.times.10.sup.6/mL) in 3-liter Fenwall culture bags in AimV+additives and IL-2 (no G418). Cell numbers were adjusted to 1.times.10.sup.6/mL, and IL-2 was added every 2 days, until sufficient numbers for treatment were achieved.

[0057] The present invention relates to a strategy of adoptive cell transfer of monocytes or DC transduced to express a chimeric antigen receptor (CAR). CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., tumor endothelial antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor endothelium cellular immune activity. In one embodiment the present invention relates generally to the use of monocytes or DC cells genetically modified to stably express a desired CAR that possesses high affinity towards tumor associated endothelium. Monocytes or DC cells expressing a CAR are referred to herein as CAR-DC cells or CAR modified DC cells. Preferably, the cell can be genetically modified to stably express an antibody binding domain on its surface, conferring novel antigen specificity that is MHC independent. In some instances, the monocyte or DC cell is genetically modified to stably express a CAR that combines an antigen recognition domain of a specific antibody with an intracellular domain of the CD3-zeta chain or Fc.gamma.RI protein into a single chimeric protein. In another embodiment, TLR signaling molecules are engineered in the intracellular portion of the CAR, said molecules include TRIF, TRADD, and MyD99. In one embodiment, the CAR of the invention comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. Preferably, the transmembrane domain is the CD8.alpha. hinge domain.

[0058] With respect to the cytoplasmic domain, the CAR of the invention can be designed to comprise the CD80 and/or CD86 and/or CD40L and/or OX40L signaling domain by itself or be combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. In one embodiment, the cytoplasmic domain of the CAR can be designed to further comprise the signaling domain of MyD88. For example, the cytoplasmic domain of the CAR can include but is not limited to CD80 and/or CD88 and/or CD40L and/or OX40L signaling modules and combinations thereof. In another embodiment of the invention inhibition of TGF-beta is performed either by transfection with an shRNA possessing selectively towards TGF-beta or by constructing the CAR to possess a dominant negative mutant of TGF-beta receptor. This would render the CAR-DC cell resistant to inhibitory activities of the tumors. Accordingly, the invention provides CAR-DC cells and methods of their use for adoptive therapy. In one embodiment, the CAR-DC cells of the invention can be generated by introducing a lentiviral vector comprising a desired CAR, for example a CAR comprising anti-CD19, CD8.alpha. hinge and transmembrane domain, and MyD88, into the cells. The CAR-DC cells of the invention are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.

[0059] One skilled in the art will appreciate that these methods, compositions, and cells are and may be adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods, procedures, and devices described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the disclosure. It will be apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Those skilled in the art recognize that the aspects and embodiments of the invention set forth herein may be practiced separate from each other or in conjunction with each other. Therefore, combinations of separate embodiments are within the scope of the invention as disclosed herein. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0060] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions indicates the exclusion of equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention disclosed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the disclosure.

Sequence CWU 1

1

391757PRTHuman 1Met Leu Leu Arg Leu Leu Leu Ala Trp Ala Ala Ala Gly Pro Thr Leu1 5 10 15Gly Gln Asp Pro Trp Ala Ala Glu Pro Arg Ala Ala Cys Gly Pro Ser 20 25 30Ser Cys Tyr Ala Leu Phe Pro Arg Arg Arg Thr Phe Leu Glu Ala Trp 35 40 45Arg Ala Cys Arg Glu Leu Gly Gly Asp Leu Ala Thr Pro Arg Thr Pro 50 55 60Glu Glu Ala Gln Arg Val Asp Ser Leu Val Gly Ala Gly Pro Ala Ser65 70 75 80Arg Leu Leu Trp Ile Gly Leu Gln Arg Gln Ala Arg Gln Cys Gln Leu 85 90 95Gln Arg Pro Leu Arg Gly Phe Thr Trp Thr Thr Gly Asp Gln Asp Thr 100 105 110Ala Phe Thr Asn Trp Ala Gln Pro Ala Ser Gly Gly Pro Cys Pro Ala 115 120 125Gln Arg Cys Val Ala Leu Glu Ala Ser Gly Glu His Arg Trp Leu Glu 130 135 140Gly Ser Cys Thr Leu Ala Val Asp Gly Tyr Leu Cys Gln Phe Gly Phe145 150 155 160Glu Gly Ala Cys Pro Ala Leu Gln Asp Glu Ala Gly Gln Ala Gly Pro 165 170 175Ala Val Tyr Thr Thr Pro Phe His Leu Val Ser Thr Glu Phe Glu Trp 180 185 190Leu Pro Phe Gly Ser Val Ala Ala Val Gln Cys Gln Ala Gly Arg Gly 195 200 205Ala Ser Leu Leu Cys Val Lys Gln Pro Glu Gly Gly Val Gly Trp Ser 210 215 220Arg Ala Gly Pro Leu Cys Leu Gly Thr Gly Cys Ser Pro Asp Asn Gly225 230 235 240Gly Cys Glu His Glu Cys Val Glu Glu Val Asp Gly His Val Ser Cys 245 250 255Arg Cys Thr Glu Gly Phe Arg Leu Ala Ala Asp Gly Arg Ser Cys Glu 260 265 270Asp Pro Cys Ala Gln Ala Pro Cys Glu Gln Gln Cys Glu Pro Gly Gly 275 280 285Pro Gln Gly Tyr Ser Cys His Cys Arg Leu Gly Phe Arg Pro Ala Glu 290 295 300Asp Asp Pro His Arg Cys Val Asp Thr Asp Glu Cys Gln Ile Ala Gly305 310 315 320Val Cys Gln Gln Met Cys Val Asn Tyr Val Gly Gly Phe Glu Cys Tyr 325 330 335Cys Ser Glu Gly His Glu Leu Glu Ala Asp Gly Ile Ser Cys Ser Pro 340 345 350Ala Gly Ala Met Gly Ala Gln Ala Ser Gln Asp Leu Gly Asp Glu Leu 355 360 365Leu Asp Asp Gly Glu Asp Glu Glu Asp Glu Asp Glu Ala Trp Lys Ala 370 375 380Phe Asn Gly Gly Trp Thr Glu Met Pro Gly Ile Leu Trp Met Glu Pro385 390 395 400Thr Gln Pro Pro Asp Phe Ala Leu Ala Tyr Arg Pro Ser Phe Pro Glu 405 410 415Asp Arg Glu Pro Gln Ile Pro Tyr Pro Glu Pro Thr Trp Pro Pro Pro 420 425 430Leu Ser Ala Pro Arg Val Pro Tyr His Ser Ser Val Leu Ser Val Thr 435 440 445Arg Pro Val Val Val Ser Ala Thr His Pro Thr Leu Pro Ser Ala His 450 455 460Gln Pro Pro Val Ile Pro Ala Thr His Pro Ala Leu Ser Arg Asp His465 470 475 480Gln Ile Pro Val Ile Ala Ala Asn Tyr Pro Asp Leu Pro Ser Ala Tyr 485 490 495Gln Pro Gly Ile Leu Ser Val Ser His Ser Ala Gln Pro Pro Ala His 500 505 510Gln Pro Pro Met Ile Ser Thr Lys Tyr Pro Glu Leu Phe Pro Ala His 515 520 525Gln Ser Pro Met Phe Pro Asp Thr Arg Val Ala Gly Thr Gln Thr Thr 530 535 540Thr His Leu Pro Gly Ile Pro Pro Asn His Ala Pro Leu Val Thr Thr545 550 555 560Leu Gly Ala Gln Leu Pro Pro Gln Ala Pro Asp Ala Leu Val Leu Arg 565 570 575Thr Gln Ala Thr Gln Leu Pro Ile Ile Pro Thr Ala Gln Pro Ser Leu 580 585 590Thr Thr Thr Ser Arg Ser Pro Val Ser Pro Ala His Gln Ile Ser Val 595 600 605Pro Ala Ala Thr Gln Pro Ala Ala Leu Pro Thr Leu Leu Pro Ser Gln 610 615 620Ser Pro Thr Asn Gln Thr Ser Pro Ile Ser Pro Thr His Pro His Ser625 630 635 640Lys Ala Pro Gln Ile Pro Arg Glu Asp Gly Pro Ser Pro Lys Leu Ala 645 650 655Leu Trp Leu Pro Ser Pro Ala Pro Thr Ala Ala Pro Thr Ala Leu Gly 660 665 670Glu Ala Gly Leu Ala Glu His Ser Gln Arg Asp Asp Arg Trp Leu Leu 675 680 685Val Ala Leu Leu Val Pro Thr Cys Val Phe Leu Val Val Leu Leu Ala 690 695 700Leu Gly Ile Val Tyr Cys Thr Arg Cys Gly Pro His Ala Pro Asn Lys705 710 715 720Arg Ile Thr Asp Cys Tyr Arg Trp Val Ile His Ala Gly Ser Lys Ser 725 730 735Pro Thr Glu Pro Met Pro Pro Arg Gly Ser Leu Thr Gly Val Gln Thr 740 745 750Cys Arg Thr Ser Val 75521443PRTHuman 2Met Ser Leu Leu Met Phe Thr Gln Leu Leu Leu Cys Gly Phe Leu Tyr1 5 10 15Val Arg Val Asp Gly Ser Arg Leu Arg Gln Glu Asp Phe Pro Pro Arg 20 25 30Ile Val Glu His Pro Ser Asp Val Ile Val Ser Lys Gly Glu Pro Thr 35 40 45Thr Leu Asn Cys Lys Ala Glu Gly Arg Pro Thr Pro Thr Ile Glu Trp 50 55 60Tyr Lys Asp Gly Glu Arg Val Glu Thr Asp Lys Asp Asp Pro Arg Ser65 70 75 80His Arg Met Leu Leu Pro Ser Gly Ser Leu Phe Phe Leu Arg Ile Val 85 90 95His Gly Arg Arg Ser Lys Pro Asp Glu Gly Ser Tyr Val Cys Val Ala 100 105 110Arg Asn Tyr Leu Gly Glu Ala Val Ser Arg Asn Ala Ser Leu Glu Val 115 120 125Ala Leu Leu Arg Asp Asp Phe Arg Gln Asn Pro Thr Asp Val Val Val 130 135 140Ala Ala Gly Glu Pro Ala Ile Leu Glu Cys Gln Pro Pro Arg Gly His145 150 155 160Pro Glu Pro Thr Ile Tyr Trp Lys Lys Asp Lys Val Arg Ile Asp Asp 165 170 175Lys Glu Glu Arg Ile Ser Ile Arg Gly Gly Lys Leu Met Ile Ser Asn 180 185 190Thr Arg Lys Ser Asp Ala Gly Met Tyr Thr Cys Val Gly Thr Asn Met 195 200 205Val Gly Glu Arg Asp Ser Asp Pro Ala Glu Leu Thr Val Phe Glu Arg 210 215 220Pro Thr Phe Leu Arg Arg Pro Ile Asn Gln Val Val Leu Glu Glu Glu225 230 235 240Ala Val Glu Phe Arg Cys Gln Val Gln Gly Asp Pro Gln Pro Thr Val 245 250 255Arg Trp Lys Lys Asp Asp Ala Asp Leu Pro Arg Gly Arg Tyr Asp Ile 260 265 270Lys Asp Asp Tyr Thr Leu Arg Ile Lys Lys Thr Met Ser Thr Asp Glu 275 280 285Gly Thr Tyr Met Cys Ile Ala Glu Asn Arg Val Gly Lys Met Glu Ala 290 295 300Ser Ala Thr Leu Thr Val Arg Ala Arg Pro Val Ala Pro Pro Gln Phe305 310 315 320Val Val Arg Pro Arg Asp Gln Ile Val Ala Gln Gly Arg Thr Val Thr 325 330 335Phe Pro Cys Glu Thr Lys Gly Asn Pro Gln Pro Ala Val Phe Trp Gln 340 345 350Lys Glu Gly Ser Gln Asn Leu Leu Phe Pro Asn Gln Pro Gln Gln Pro 355 360 365Asn Ser Arg Cys Ser Val Ser Pro Thr Gly Asp Leu Thr Ile Thr Asn 370 375 380Ile Gln Arg Ser Asp Ala Gly Tyr Tyr Ile Cys Gln Ala Leu Thr Val385 390 395 400Ala Gly Ser Ile Leu Ala Lys Ala Gln Leu Glu Val Thr Asp Val Leu 405 410 415Thr Asp Arg Pro Pro Pro Ile Ile Leu Gln Gly Pro Ala Asn Gln Thr 420 425 430Leu Ala Val Asp Gly Thr Ala Leu Leu Lys Cys Lys Ala Thr Gly Asp 435 440 445Pro Leu Pro Val Ile Ser Trp Leu Lys Glu Gly Phe Thr Phe Pro Gly 450 455 460Arg Asp Pro Arg Ala Thr Ile Gln Glu Gln Gly Thr Leu Gln Ile Lys465 470 475 480Asn Leu Arg Ile Ser Asp Thr Gly Thr Tyr Thr Cys Val Ala Thr Ser 485 490 495Ser Ser Gly Glu Thr Ser Trp Ser Ala Val Leu Asp Val Thr Glu Ser 500 505 510Gly Ala Thr Ile Ser Lys Asn Tyr Asp Leu Ser Asp Leu Pro Gly Pro 515 520 525Pro Ser Lys Pro Gln Val Thr Asp Val Thr Lys Asn Ser Val Thr Leu 530 535 540Ser Trp Gln Pro Gly Thr Pro Gly Thr Leu Pro Ala Ser Ala Tyr Ile545 550 555 560Ile Glu Ala Phe Ser Gln Ser Val Ser Asn Ser Trp Gln Thr Val Ala 565 570 575Asn His Val Lys Thr Thr Leu Tyr Thr Val Arg Gly Leu Arg Pro Asn 580 585 590Thr Ile Tyr Leu Phe Met Val Arg Ala Ile Asn Pro Gln Gly Leu Ser 595 600 605Asp Pro Ser Pro Met Ser Asp Pro Val Arg Thr Gln Asp Ile Ser Pro 610 615 620Pro Ala Gln Gly Val Asp His Arg Gln Val Gln Lys Glu Leu Gly Asp625 630 635 640Val Leu Val Arg Leu His Asn Pro Val Val Leu Thr Pro Thr Thr Val 645 650 655Gln Val Thr Trp Thr Val Asp Arg Gln Pro Gln Phe Ile Gln Gly Tyr 660 665 670Arg Val Met Tyr Arg Gln Thr Ser Gly Leu Gln Ala Thr Ser Ser Trp 675 680 685Gln Asn Leu Asp Ala Lys Val Pro Thr Glu Arg Ser Ala Val Leu Val 690 695 700Asn Leu Lys Lys Gly Val Thr Tyr Glu Ile Lys Val Arg Pro Tyr Phe705 710 715 720Asn Glu Phe Gln Gly Met Asp Ser Glu Ser Lys Thr Val Arg Thr Thr 725 730 735Glu Glu Ala Pro Ser Ala Pro Pro Gln Ser Val Thr Val Leu Thr Val 740 745 750Gly Ser Tyr Asn Ser Thr Ser Ile Ser Val Ser Trp Asp Pro Pro Pro 755 760 765Pro Asp His Gln Asn Gly Ile Ile Gln Glu Tyr Lys Ile Trp Cys Leu 770 775 780Gly Asn Glu Thr Arg Phe His Ile Asn Lys Thr Val Asp Ala Ala Ile785 790 795 800Arg Ser Val Ile Ile Gly Gly Leu Phe Pro Gly Ile Gln Tyr Arg Val 805 810 815Glu Val Ala Ala Ser Thr Ser Ala Gly Val Gly Val Lys Ser Glu Pro 820 825 830Gln Pro Ile Ile Ile Gly Arg Arg Asn Glu Val Val Ile Thr Glu Asn 835 840 845Asn Asn Ser Ile Thr Glu Gln Ile Thr Asp Val Val Lys Gln Pro Ala 850 855 860Phe Ile Ala Gly Ile Gly Gly Ala Cys Trp Val Ile Leu Met Gly Phe865 870 875 880Ser Ile Trp Leu Tyr Trp Arg Arg Lys Lys Arg Lys Gly Leu Ser Asn 885 890 895Tyr Ala Val Thr Phe Gln Arg Gly Asp Gly Gly Leu Met Ser Asn Gly 900 905 910Ser Arg Pro Gly Leu Leu Asn Ala Gly Asp Pro Ser Tyr Pro Trp Leu 915 920 925Ala Asp Ser Trp Pro Ala Thr Ser Leu Pro Val Asn Asn Ser Asn Ser 930 935 940Gly Pro Asn Glu Ile Gly Asn Phe Gly Arg Gly Asp Val Leu Pro Pro945 950 955 960Val Pro Gly Gln Gly Asp Lys Thr Ala Thr Met Leu Ser Asp Gly Ala 965 970 975Ile Tyr Ser Ser Ile Asp Phe Thr Thr Lys Thr Ser Tyr Asn Ser Ser 980 985 990Ser Gln Ile Thr Gln Ala Thr Pro Tyr Ala Thr Thr Gln Ile Leu His 995 1000 1005Ser Asn Ser Ile His Glu Leu Ala Val Asp Leu Pro Asp Pro Gln 1010 1015 1020Trp Lys Ser Ser Ile Gln Gln Lys Thr Asp Leu Met Gly Phe Gly 1025 1030 1035Tyr Ser Leu Pro Asp Gln Asn Lys Gly Asn Asn Gly Gly Lys Gly 1040 1045 1050Gly Lys Lys Lys Lys Asn Lys Asn Ser Ser Lys Pro Gln Lys Asn 1055 1060 1065Asn Gly Ser Thr Trp Ala Asn Val Pro Leu Pro Pro Pro Pro Val 1070 1075 1080Gln Pro Leu Pro Gly Thr Glu Leu Glu His Tyr Ala Val Glu Gln 1085 1090 1095Gln Glu Asn Gly Tyr Asp Ser Asp Ser Trp Cys Pro Pro Leu Pro 1100 1105 1110Val Gln Thr Tyr Leu His Gln Gly Leu Glu Asp Glu Leu Glu Glu 1115 1120 1125Asp Asp Asp Arg Val Pro Thr Pro Pro Val Arg Gly Val Ala Ser 1130 1135 1140Ser Pro Ala Ile Ser Phe Gly Gln Gln Ser Thr Ala Thr Leu Thr 1145 1150 1155Pro Ser Pro Arg Glu Glu Met Gln Pro Met Leu Gln Ala His Leu 1160 1165 1170Asp Glu Leu Thr Arg Ala Tyr Gln Phe Asp Ile Ala Lys Gln Thr 1175 1180 1185Trp His Ile Gln Ser Asn Asn Gln Pro Pro Gln Pro Pro Val Pro 1190 1195 1200Pro Leu Gly Tyr Val Ser Gly Ala Leu Ile Ser Asp Leu Glu Thr 1205 1210 1215Asp Val Ala Asp Asp Asp Ala Asp Asp Glu Glu Glu Ala Leu Glu 1220 1225 1230Ile Pro Arg Pro Leu Arg Ala Leu Asp Gln Thr Pro Gly Ser Ser 1235 1240 1245Met Asp Asn Leu Asp Ser Ser Val Thr Gly Ser Met Val Asn Gly 1250 1255 1260Trp Gly Ser Ala Ser Asp Glu Asp Arg Asn Phe Ser Ser His Arg 1265 1270 1275Ser Ser Val Gly Ser Ser Ser Asp Gly Ser Ile Phe Ala Ser Gly 1280 1285 1290Ser Phe Ala Gln Ala Leu Val Ala Ala Ala Asp Lys Ala Gly Phe 1295 1300 1305Arg Leu Asp Gly Thr Ser Leu Thr Arg Thr Gly Lys Ala Phe Thr 1310 1315 1320Ser Ser Gln Arg Pro Arg Pro Thr Ser Pro Phe Ser Thr Asp Ser 1325 1330 1335Asn Thr Ser Ala Ala Leu Ser Gln Ser Gln Arg Pro Arg Pro Thr 1340 1345 1350Lys Lys His Lys Gly Gly Arg Met Asp Gln Gln Pro Ala Leu Pro 1355 1360 1365His Arg Arg Glu Gly Met Thr Asp Glu Glu Ala Leu Val Pro Tyr 1370 1375 1380Ser Lys Pro Ser Phe Pro Ser Pro Gly Gly His Ser Ser Ser Gly 1385 1390 1395Thr Ala Ser Ser Lys Gly Ser Thr Gly Pro Arg Lys Thr Glu Val 1400 1405 1410Leu Arg Ala Gly His Gln Arg Asn Ala Ser Asp Leu Leu Asp Ile 1415 1420 1425Gly Tyr Met Gly Ser Asn Ser Gln Gly Gln Phe Thr Gly Glu Leu 1430 1435 14403712PRTHuman 3Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu1 5 10 15Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro 20 25 30Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr 35 40 45Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro 50 55 60Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser65 70 75 80Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn 85 90 95Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser 100 105 110Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser 115 120 125Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys 130 135 140Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser145 150 155 160Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg 165 170 175Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile 180 185 190Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser 195 200 205Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr 210 215 220Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu225 230 235 240Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile 245 250 255Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

His Gln His Lys Lys Leu 260 265 270Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe 275 280 285Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu 290 295 300Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr305 310 315 320Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met 325 330 335Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala 340 345 350Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly 355 360 365Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr 370 375 380Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu385 390 395 400Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val 405 410 415Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val 420 425 430Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr 435 440 445Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu 450 455 460Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr465 470 475 480Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys 485 490 495Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys 500 505 510Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr 515 520 525Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser 530 535 540Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln545 550 555 560Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser 565 570 575Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro 580 585 590Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr 595 600 605Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile 610 615 620Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr625 630 635 640Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val 645 650 655Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn 660 665 670Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys 675 680 685Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn 690 695 700Glu Thr Leu Val Glu Asp Ser Glu705 7104658PRTHuman 4Met Asp Arg Gly Thr Leu Pro Leu Ala Val Ala Leu Leu Leu Ala Ser1 5 10 15Cys Ser Leu Ser Pro Thr Ser Leu Ala Glu Thr Val His Cys Asp Leu 20 25 30Gln Pro Val Gly Pro Glu Arg Gly Glu Val Thr Tyr Thr Thr Ser Gln 35 40 45Val Ser Lys Gly Cys Val Ala Gln Ala Pro Asn Ala Ile Leu Glu Val 50 55 60His Val Leu Phe Leu Glu Phe Pro Thr Gly Pro Ser Gln Leu Glu Leu65 70 75 80Thr Leu Gln Ala Ser Lys Gln Asn Gly Thr Trp Pro Arg Glu Val Leu 85 90 95Leu Val Leu Ser Val Asn Ser Ser Val Phe Leu His Leu Gln Ala Leu 100 105 110Gly Ile Pro Leu His Leu Ala Tyr Asn Ser Ser Leu Val Thr Phe Gln 115 120 125Glu Pro Pro Gly Val Asn Thr Thr Glu Leu Pro Ser Phe Pro Lys Thr 130 135 140Gln Ile Leu Glu Trp Ala Ala Glu Arg Gly Pro Ile Thr Ser Ala Ala145 150 155 160Glu Leu Asn Asp Pro Gln Ser Ile Leu Leu Arg Leu Gly Gln Ala Gln 165 170 175Gly Ser Leu Ser Phe Cys Met Leu Glu Ala Ser Gln Asp Met Gly Arg 180 185 190Thr Leu Glu Trp Arg Pro Arg Thr Pro Ala Leu Val Arg Gly Cys His 195 200 205Leu Glu Gly Val Ala Gly His Lys Glu Ala His Ile Leu Arg Val Leu 210 215 220Pro Gly His Ser Ala Gly Pro Arg Thr Val Thr Val Lys Val Glu Leu225 230 235 240Ser Cys Ala Pro Gly Asp Leu Asp Ala Val Leu Ile Leu Gln Gly Pro 245 250 255Pro Tyr Val Ser Trp Leu Ile Asp Ala Asn His Asn Met Gln Ile Trp 260 265 270Thr Thr Gly Glu Tyr Ser Phe Lys Ile Phe Pro Glu Lys Asn Ile Arg 275 280 285Gly Phe Lys Leu Pro Asp Thr Pro Gln Gly Leu Leu Gly Glu Ala Arg 290 295 300Met Leu Asn Ala Ser Ile Val Ala Ser Phe Val Glu Leu Pro Leu Ala305 310 315 320Ser Ile Val Ser Leu His Ala Ser Ser Cys Gly Gly Arg Leu Gln Thr 325 330 335Ser Pro Ala Pro Ile Gln Thr Thr Pro Pro Lys Asp Thr Cys Ser Pro 340 345 350Glu Leu Leu Met Ser Leu Ile Gln Thr Lys Cys Ala Asp Asp Ala Met 355 360 365Thr Leu Val Leu Lys Lys Glu Leu Val Ala His Leu Lys Cys Thr Ile 370 375 380Thr Gly Leu Thr Phe Trp Asp Pro Ser Cys Glu Ala Glu Asp Arg Gly385 390 395 400Asp Lys Phe Val Leu Arg Ser Ala Tyr Ser Ser Cys Gly Met Gln Val 405 410 415Ser Ala Ser Met Ile Ser Asn Glu Ala Val Val Asn Ile Leu Ser Ser 420 425 430Ser Ser Pro Gln Arg Lys Lys Val His Cys Leu Asn Met Asp Ser Leu 435 440 445Ser Phe Gln Leu Gly Leu Tyr Leu Ser Pro His Phe Leu Gln Ala Ser 450 455 460Asn Thr Ile Glu Pro Gly Gln Gln Ser Phe Val Gln Val Arg Val Ser465 470 475 480Pro Ser Val Ser Glu Phe Leu Leu Gln Leu Asp Ser Cys His Leu Asp 485 490 495Leu Gly Pro Glu Gly Gly Thr Val Glu Leu Ile Gln Gly Arg Ala Ala 500 505 510Lys Gly Asn Cys Val Ser Leu Leu Ser Pro Ser Pro Glu Gly Asp Pro 515 520 525Arg Phe Ser Phe Leu Leu His Phe Tyr Thr Val Pro Ile Pro Lys Thr 530 535 540Gly Thr Leu Ser Cys Thr Val Ala Leu Arg Pro Lys Thr Gly Ser Gln545 550 555 560Asp Gln Glu Val His Arg Thr Val Phe Met Arg Leu Asn Ile Ile Ser 565 570 575Pro Asp Leu Ser Gly Cys Thr Ser Lys Gly Leu Val Leu Pro Ala Val 580 585 590Leu Gly Ile Thr Phe Gly Ala Phe Leu Ile Gly Ala Leu Leu Thr Ala 595 600 605Ala Leu Trp Tyr Ile Tyr Ser His Thr Arg Ser Pro Ser Lys Arg Glu 610 615 620Pro Val Val Ala Val Ala Ala Pro Ala Ser Ser Glu Ser Ser Ser Thr625 630 635 640Asn His Ser Ile Gly Ser Thr Gln Ser Thr Pro Cys Ser Thr Ser Ser 645 650 655Met Ala5117PRTHuman 5Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp1 5 10 15His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala 20 25 30Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr 35 40 45Glu Asn Glu Pro Asp Leu Ala Gln Trp Val Phe Cys Phe Lys Glu Leu 50 55 60Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys His65 70 75 80Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu 85 90 95Thr Leu Gly Glu Phe Leu Lys Leu Val Arg Glu Thr Leu Pro Pro Pro 100 105 110Arg Ser Phe Ile Arg 1156652PRTHuman 6Met Ala Thr Ser Met Gly Leu Leu Leu Leu Leu Leu Leu Leu Leu Thr1 5 10 15Gln Pro Gly Ala Gly Thr Gly Ala Asp Thr Glu Ala Val Val Cys Val 20 25 30Gly Thr Ala Cys Tyr Thr Ala His Ser Gly Lys Leu Ser Ala Ala Glu 35 40 45Ala Gln Asn His Cys Asn Gln Asn Gly Gly Asn Leu Ala Thr Val Lys 50 55 60Ser Lys Glu Glu Ala Gln His Val Gln Arg Val Leu Ala Gln Leu Leu65 70 75 80Arg Arg Glu Ala Ala Leu Thr Ala Arg Met Ser Lys Phe Trp Ile Gly 85 90 95Leu Gln Arg Glu Lys Gly Lys Cys Leu Asp Pro Ser Leu Pro Leu Lys 100 105 110Gly Phe Ser Trp Val Gly Gly Gly Glu Asp Thr Pro Tyr Ser Asn Trp 115 120 125His Lys Glu Leu Arg Asn Ser Cys Ile Ser Lys Arg Cys Val Ser Leu 130 135 140Leu Leu Asp Leu Ser Gln Pro Leu Leu Pro Ser Arg Leu Pro Lys Trp145 150 155 160Ser Glu Gly Pro Cys Gly Ser Pro Gly Ser Pro Gly Ser Asn Ile Glu 165 170 175Gly Phe Val Cys Lys Phe Ser Phe Lys Gly Met Cys Arg Pro Leu Ala 180 185 190Leu Gly Gly Pro Gly Gln Val Thr Tyr Thr Thr Pro Phe Gln Thr Thr 195 200 205Ser Ser Ser Leu Glu Ala Val Pro Phe Ala Ser Ala Ala Asn Val Ala 210 215 220Cys Gly Glu Gly Asp Lys Asp Glu Thr Gln Ser His Tyr Phe Leu Cys225 230 235 240Lys Glu Lys Ala Pro Asp Val Phe Asp Trp Gly Ser Ser Gly Pro Leu 245 250 255Cys Val Ser Pro Lys Tyr Gly Cys Asn Phe Asn Asn Gly Gly Cys His 260 265 270Gln Asp Cys Phe Glu Gly Gly Asp Gly Ser Phe Leu Cys Gly Cys Arg 275 280 285Pro Gly Phe Arg Leu Leu Asp Asp Leu Val Thr Cys Ala Ser Arg Asn 290 295 300Pro Cys Ser Ser Ser Pro Cys Arg Gly Gly Ala Thr Cys Val Leu Gly305 310 315 320Pro His Gly Lys Asn Tyr Thr Cys Arg Cys Pro Gln Gly Tyr Gln Leu 325 330 335Asp Ser Ser Gln Leu Asp Cys Val Asp Val Asp Glu Cys Gln Asp Ser 340 345 350Pro Cys Ala Gln Glu Cys Val Asn Thr Pro Gly Gly Phe Arg Cys Glu 355 360 365Cys Trp Val Gly Tyr Glu Pro Gly Gly Pro Gly Glu Gly Ala Cys Gln 370 375 380Asp Val Asp Glu Cys Ala Leu Gly Arg Ser Pro Cys Ala Gln Gly Cys385 390 395 400Thr Asn Thr Asp Gly Ser Phe His Cys Ser Cys Glu Glu Gly Tyr Val 405 410 415Leu Ala Gly Glu Asp Gly Thr Gln Cys Gln Asp Val Asp Glu Cys Val 420 425 430Gly Pro Gly Gly Pro Leu Cys Asp Ser Leu Cys Phe Asn Thr Gln Gly 435 440 445Ser Phe His Cys Gly Cys Leu Pro Gly Trp Val Leu Ala Pro Asn Gly 450 455 460Val Ser Cys Thr Met Gly Pro Val Ser Leu Gly Pro Pro Ser Gly Pro465 470 475 480Pro Asp Glu Glu Asp Lys Gly Glu Lys Glu Gly Ser Thr Val Pro Arg 485 490 495Ala Ala Thr Ala Ser Pro Thr Arg Gly Pro Glu Gly Thr Pro Lys Ala 500 505 510Thr Pro Thr Thr Ser Arg Pro Ser Leu Ser Ser Asp Ala Pro Ile Thr 515 520 525Ser Ala Pro Leu Lys Met Leu Ala Pro Ser Gly Ser Ser Gly Val Trp 530 535 540Arg Glu Pro Ser Ile His His Ala Thr Ala Ala Ser Gly Pro Gln Glu545 550 555 560Pro Ala Gly Gly Asp Ser Ser Val Ala Thr Gln Asn Asn Asp Gly Thr 565 570 575Asp Gly Gln Lys Leu Leu Leu Phe Tyr Ile Leu Gly Thr Val Val Ala 580 585 590Ile Leu Leu Leu Leu Ala Leu Ala Leu Gly Leu Leu Val Tyr Arg Lys 595 600 605Arg Arg Ala Lys Arg Glu Glu Lys Lys Glu Lys Lys Pro Gln Asn Ala 610 615 620Ala Asp Ser Tyr Ser Trp Val Pro Glu Arg Ala Glu Ser Arg Ala Met625 630 635 640Glu Asn Gln Tyr Ser Pro Thr Pro Gly Thr Asp Cys 645 6507277PRTHuman 7Met Trp Pro Gly Ile Leu Val Gly Gly Ala Arg Val Ala Ser Cys Arg1 5 10 15Tyr Pro Ala Leu Gly Pro Arg Leu Ala Ala His Phe Pro Ala Gln Arg 20 25 30Pro Pro Gln Arg Thr Leu Gln Asn Gly Leu Ala Leu Gln Arg Cys Leu 35 40 45His Ala Thr Ala Thr Arg Ala Leu Pro Leu Ile Pro Ile Val Val Glu 50 55 60Gln Thr Gly Arg Gly Glu Arg Ala Tyr Asp Ile Tyr Ser Arg Leu Leu65 70 75 80Arg Glu Arg Ile Val Cys Val Met Gly Pro Ile Asp Asp Ser Val Ala 85 90 95Ser Leu Val Ile Ala Gln Leu Leu Phe Leu Gln Ser Glu Ser Asn Lys 100 105 110Lys Pro Ile His Met Tyr Ile Asn Ser Pro Gly Gly Val Val Thr Ala 115 120 125Gly Leu Ala Ile Tyr Asp Thr Met Gln Tyr Ile Leu Asn Pro Ile Cys 130 135 140Thr Trp Cys Val Gly Gln Ala Ala Ser Met Gly Ser Leu Leu Leu Ala145 150 155 160Ala Gly Thr Pro Gly Met Arg His Ser Leu Pro Asn Ser Arg Ile Met 165 170 175Ile His Gln Pro Ser Gly Gly Ala Arg Gly Gln Ala Thr Asp Ile Ala 180 185 190Ile Gln Ala Glu Glu Ile Met Lys Leu Lys Lys Gln Leu Tyr Asn Ile 195 200 205Tyr Ala Lys His Thr Lys Gln Ser Leu Gln Val Ile Glu Ser Ala Met 210 215 220Glu Arg Asp Arg Tyr Met Ser Pro Met Glu Ala Gln Glu Phe Gly Ile225 230 235 240Leu Asp Lys Val Leu Val His Pro Pro Gln Asp Gly Glu Asp Glu Pro 245 250 255Thr Leu Val Gln Lys Glu Pro Val Glu Ala Ala Pro Ala Ala Glu Pro 260 265 270Val Pro Ala Ser Thr 2758285PRTHuman 8Met Ala Ala Ala Glu Ala Ala Asn Cys Ile Met Glu Val Ser Cys Gly1 5 10 15Gln Ala Glu Ser Ser Glu Lys Pro Asn Ala Glu Asp Met Thr Ser Lys 20 25 30Asp Tyr Tyr Phe Asp Ser Tyr Ala His Phe Gly Ile His Glu Glu Met 35 40 45Leu Lys Asp Glu Val Arg Thr Leu Thr Tyr Arg Asn Ser Met Phe His 50 55 60Asn Arg His Leu Phe Lys Asp Lys Val Val Leu Asp Val Gly Ser Gly65 70 75 80Thr Gly Ile Leu Cys Met Phe Ala Ala Lys Ala Gly Ala Arg Lys Val 85 90 95Ile Gly Ile Glu Cys Ser Ser Ile Ser Asp Tyr Ala Val Lys Ile Val 100 105 110Lys Ala Asn Lys Leu Asp His Val Val Thr Ile Ile Lys Gly Lys Val 115 120 125Glu Glu Val Glu Leu Pro Val Glu Lys Val Asp Ile Ile Ile Ser Glu 130 135 140Trp Met Gly Tyr Cys Leu Phe Tyr Glu Ser Met Leu Asn Thr Val Leu145 150 155 160Tyr Ala Arg Asp Lys Trp Leu Glu Val Asp Ile Tyr Thr Val Lys Val 165 170 175Glu Asp Leu Thr Phe Thr Ser Pro Phe Cys Leu Gln Val Lys Arg Asn 180 185 190Asp Tyr Val His Ala Leu Val Ala Tyr Phe Asn Ile Glu Phe Thr Arg 195 200 205Cys His Lys Arg Thr Gly Phe Ser Thr Ser Pro Glu Ser Pro Tyr Thr 210 215 220His Trp Lys Gln Thr Val Phe Tyr Met Glu Asp Tyr Leu Thr Val Lys225 230 235 240Thr Gly Glu Glu Ile Phe Gly Thr Ile Gly Met Arg Pro Asn Ala Lys 245 250 255Asn Asn Arg Asp Leu Asp Phe Thr Ile Asp Leu Asp Phe Lys Gly Gln 260 265 270Leu Cys Glu Leu Ser Cys Ser Thr Asp Tyr Arg Met Arg 275 280 2859609PRTHuman 9Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr1 5 10 15Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu 20

25 30Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr 35 40 45Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser 50 55 60Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp65 70 75 80Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu 85 90 95Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp 100 105 110Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His 115 120 125Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro 130 135 140Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn145 150 155 160Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro 165 170 175Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys 180 185 190Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr 195 200 205Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys 210 215 220Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val225 230 235 240Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln 245 250 255Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly 260 265 270Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile 275 280 285Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys 290 295 300Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp305 310 315 320Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp 325 330 335Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala 340 345 350Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser 355 360 365Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys 370 375 380Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu385 390 395 400Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys 405 410 415Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu 420 425 430Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met 435 440 445Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu 450 455 460Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile465 470 475 480Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly 485 490 495Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe 500 505 510Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp 515 520 525Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala 530 535 540Leu Gln Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys545 550 555 560Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser 565 570 575Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe 580 585 590Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Arg Ala Ala Leu Gly 595 600 605Val1043PRTHuman 10Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln1 5 10 15Ala Arg Leu Met Lys Glu Glu Ser Pro Val Val Ser Trp Arg Leu Glu 20 25 30Pro Glu Asp Gly Thr Ala Leu Cys Phe Ile Phe 35 4011198PRTHuman 11Leu Val Ser Glu Val Ile Arg Phe Ile Leu Phe Lys Phe His Gln Ser1 5 10 15Ser Gly Thr Pro Ile Lys Arg Glu Asp Leu Thr Gln Ile Val Thr Lys 20 25 30Asn Tyr Arg Gln Arg Asn Leu Ala Thr His Val Ile Asn Glu Ala Lys 35 40 45Lys Lys Leu Ser Asn Val Phe Gly Tyr Asp Leu Lys Glu Leu Gln Arg 50 55 60Ala Arg Ala Ser Ser Thr Gly Gln Ser Arg Leu Pro Gln Ser Gln Ser65 70 75 80Ser Val Asp Ser Lys Ser Tyr Val Leu Val Ser Glu Leu Pro Leu Glu 85 90 95Val Phe Lys Lys His Val Val Asp Glu Thr Thr Ser Pro Val Thr Gly 100 105 110Phe Thr Phe Val Val Leu Ala Ile Val Gln Leu Ala Gly Gly Lys Ile 115 120 125Pro Glu Glu Thr Leu Trp His His Leu Lys Arg Met Gly Leu His Glu 130 135 140Asn Asp Glu His Asn Pro Val Phe Gly Asn Asn Lys Gln Thr Leu Glu145 150 155 160Thr Leu Val Gln Gln Arg Phe Leu Gln Lys Glu Lys Val Ser Gly Pro 165 170 175Glu Gly Ser Thr Leu Val Tyr Asp Leu Ala Glu Arg Ala Leu Asp Pro 180 185 190Gln Val Ser Glu Lys Val 19512562PRTHuman 12Met Ser Asn Gln Tyr Gln Glu Glu Gly Cys Ser Glu Arg Pro Glu Cys1 5 10 15Lys Ser Lys Ser Pro Thr Leu Leu Ser Ser Tyr Cys Ile Asp Ser Ile 20 25 30Leu Gly Arg Arg Ser Pro Cys Lys Met Arg Leu Leu Gly Ala Ala Gln 35 40 45Ser Leu Pro Ala Pro Leu Thr Ser Arg Ala Asp Pro Glu Lys Ala Val 50 55 60Gln Gly Ser Pro Lys Ser Ser Ser Ala Pro Phe Glu Ala Glu Leu His65 70 75 80Leu Pro Pro Lys Leu Arg Arg Leu Tyr Gly Pro Gly Gly Gly Arg Leu 85 90 95Leu Gln Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 100 105 110Ala Ala Ala Thr Ala Thr Ala Gly Pro Arg Gly Glu Ala Pro Pro Pro 115 120 125Pro Pro Pro Thr Ala Arg Pro Gly Glu Arg Pro Asp Gly Ala Gly Ala 130 135 140Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Trp Asp Thr Leu Lys145 150 155 160Ile Ser Gln Ala Pro Gln Val Ser Ile Ser Arg Ser Lys Ser Tyr Arg 165 170 175Glu Asn Gly Ala Pro Phe Val Pro Pro Pro Pro Ala Leu Asp Glu Leu 180 185 190Gly Gly Pro Gly Gly Val Thr His Pro Glu Glu Arg Leu Gly Val Ala 195 200 205Gly Gly Pro Gly Ser Ala Pro Ala Ala Gly Gly Gly Thr Gly Thr Glu 210 215 220Asp Asp Glu Glu Glu Leu Leu Glu Asp Glu Glu Asp Glu Asp Glu Glu225 230 235 240Glu Glu Leu Leu Glu Asp Asp Glu Glu Glu Leu Leu Glu Asp Asp Ala 245 250 255Arg Ala Leu Leu Lys Glu Pro Arg Arg Cys Pro Val Ala Ala Thr Gly 260 265 270Ala Val Ala Ala Ala Ala Ala Ala Ala Val Ala Thr Glu Gly Gly Glu 275 280 285Leu Ser Pro Lys Glu Glu Leu Leu Leu His Pro Glu Asp Ala Glu Gly 290 295 300Lys Asp Gly Glu Asp Ser Val Cys Leu Ser Ala Gly Ser Asp Ser Glu305 310 315 320Glu Gly Leu Leu Lys Arg Lys Gln Arg Arg Tyr Arg Thr Thr Phe Thr 325 330 335Ser Tyr Gln Leu Glu Glu Leu Glu Arg Ala Phe Gln Lys Thr His Tyr 340 345 350Pro Asp Val Phe Thr Arg Glu Glu Leu Ala Met Arg Leu Asp Leu Thr 355 360 365Glu Ala Arg Val Gln Val Trp Phe Gln Asn Arg Arg Ala Lys Trp Arg 370 375 380Lys Arg Glu Lys Ala Gly Ala Gln Thr His Pro Pro Gly Leu Pro Phe385 390 395 400Pro Gly Pro Leu Ser Ala Thr His Pro Leu Ser Pro Tyr Leu Asp Ala 405 410 415Ser Pro Phe Pro Pro His His Pro Ala Leu Asp Ser Ala Trp Thr Ala 420 425 430Ala Ala Ala Ala Ala Ala Ala Ala Phe Pro Ser Leu Pro Pro Pro Pro 435 440 445Gly Ser Ala Ser Leu Pro Pro Ser Gly Ala Pro Leu Gly Leu Ser Thr 450 455 460Phe Leu Gly Ala Ala Val Phe Arg His Pro Ala Phe Ile Ser Pro Ala465 470 475 480Phe Gly Arg Leu Phe Ser Thr Met Ala Pro Leu Thr Ser Ala Ser Thr 485 490 495Ala Ala Ala Leu Leu Arg Gln Pro Thr Pro Ala Val Glu Gly Ala Val 500 505 510Ala Ser Gly Ala Leu Ala Asp Pro Ala Thr Ala Ala Ala Asp Arg Arg 515 520 525Ala Ser Ser Ile Ala Ala Leu Arg Leu Lys Ala Lys Glu His Ala Ala 530 535 540Gln Leu Thr Gln Leu Asn Ile Leu Pro Gly Thr Ser Thr Gly Lys Glu545 550 555 560Val Cys13501PRTHuman 13Met Ala Pro Asp Lys Phe Leu Ser Thr Ile Thr Ala Gly Leu Met Asn1 5 10 15Phe Thr Gly Ala Asp Ile Pro Pro Leu Ser Thr Arg Asp Gln Tyr Ala 20 25 30Thr Val Asn His His Val His Glu Ala Arg Met Glu Asn Gly Gln Arg 35 40 45Lys Gln Asp Asn Val Leu Ser Asn Val Leu Ser Gly Leu Ile Asn Met 50 55 60Ala Gly Ala Ser Ile Pro Ala Met Ser Ser Arg Asp Leu Tyr Ala Thr65 70 75 80Ile Thr His Ser Val Arg Glu Glu Lys Met Glu Ser Gly Lys Pro Gln 85 90 95Thr Asp Lys Val Ile Ser Asn Asp Ala Pro Gln Leu Gly His Met Ala 100 105 110Ala Gly Gly Ile Pro Ser Met Ser Thr Lys Asp Leu Tyr Ala Thr Val 115 120 125Thr Gln Asn Val His Glu Glu Arg Met Glu Asn Asn Gln Pro Gln Pro 130 135 140Ser Tyr Asp Leu Ser Thr Val Leu Pro Gly Leu Thr Tyr Leu Thr Val145 150 155 160Ala Gly Ile Pro Ala Met Ser Thr Arg Asp Gln Tyr Ala Thr Val Thr 165 170 175His Asn Val His Glu Glu Lys Ile Lys Asn Gly Gln Ala Ala Ser Asp 180 185 190Asn Val Phe Ser Thr Val Pro Pro Ala Phe Ile Asn Met Ala Ala Thr 195 200 205Gly Val Ser Ser Met Ser Thr Arg Asp Gln Tyr Ala Ala Val Thr His 210 215 220Asn Ile Arg Glu Glu Lys Ile Asn Asn Ser Gln Pro Ala Pro Gly Asn225 230 235 240Ile Leu Ser Thr Ala Pro Pro Trp Leu Arg His Met Ala Ala Ala Gly 245 250 255Ile Ser Ser Thr Ile Thr Arg Asp Leu Tyr Val Thr Ala Thr His Ser 260 265 270Val His Glu Glu Lys Met Thr Asn Gly Gln Gln Ala Pro Asp Asn Ser 275 280 285Leu Ser Thr Val Pro Pro Gly Cys Ile Asn Leu Ser Gly Ala Gly Ile 290 295 300Ser Cys Arg Ser Thr Arg Asp Leu Tyr Ala Thr Val Ile His Asp Ile305 310 315 320Gln Glu Glu Glu Met Glu Asn Asp Gln Thr Pro Pro Asp Gly Phe Leu 325 330 335Ser Asn Ser Asp Ser Pro Glu Leu Ile Asn Met Thr Gly His Cys Met 340 345 350Pro Pro Asn Ala Leu Asp Ser Phe Ser His Asp Phe Thr Ser Leu Ser 355 360 365Lys Asp Glu Leu Leu Tyr Lys Pro Asp Ser Asn Glu Phe Ala Val Gly 370 375 380Thr Lys Asn Tyr Ser Val Ser Ala Gly Asp Pro Pro Val Thr Val Met385 390 395 400Ser Leu Val Glu Thr Val Pro Asn Thr Pro Gln Ile Ser Pro Ala Met 405 410 415Ala Lys Lys Ile Asn Asp Asp Ile Lys Tyr Gln Leu Met Lys Glu Val 420 425 430Arg Arg Phe Gly Gln Asn Tyr Glu Arg Ile Phe Ile Leu Leu Glu Glu 435 440 445Val Gln Gly Ser Met Lys Val Lys Arg Gln Phe Val Glu Phe Thr Ile 450 455 460Lys Glu Ala Ala Arg Phe Lys Lys Val Val Leu Ile Gln Gln Leu Glu465 470 475 480Lys Ala Leu Lys Glu Ile Asp Ser His Cys His Leu Arg Lys Val Lys 485 490 495His Met Arg Lys Arg 50014636PRTHuman 14Met Ser Phe Leu Ile Lys Gly Ala Gln Lys His Gly Phe Ile Cys Gln1 5 10 15Thr His Lys Glu Leu Phe Lys Arg Cys Ile Leu Lys Ser Ser Lys Thr 20 25 30Phe Asn Gly Gln Lys Met Ile Ser Ser Gln Ala Ser Ser Pro Val Asn 35 40 45Ser Lys Asn Met Asp Ser Phe Asn Tyr Ile Ile Val Gly Ala Gly Ser 50 55 60Ala Gly Cys Val Leu Ala Asn Arg Leu Thr Glu Asn Pro His Asn Thr65 70 75 80Val Lys Leu Leu Glu Ala Gly Pro Lys Asp Thr Val Leu Gly Ser Lys 85 90 95Gln Leu Ser Trp Lys Ile His Met Pro Ala Ala Leu Thr Tyr Asn Leu 100 105 110Cys Asp Glu Lys Tyr Asn Trp Tyr Tyr His Thr Thr Pro Gln Lys His 115 120 125Met Asp Asn Arg Ile Leu Tyr Trp Pro Arg Gly Arg Val Trp Gly Gly 130 135 140Ser Ser Ser Leu Asn Ala Met Val Tyr Ile Arg Gly His Ala Glu Asp145 150 155 160Tyr Asn Arg Trp Ser Lys Glu Gly Ala Val Gly Trp Asp Tyr Glu Phe 165 170 175Cys Leu Pro Tyr Phe Lys Lys Ala Gln Thr His Glu Leu Gly Ala Asp 180 185 190Leu Tyr Arg Gly Gly Asp Gly Pro Leu His Val Ser Arg Gly Lys Thr 195 200 205Lys Asn Pro Leu His Cys Ala Phe Leu Asp Ala Ala Gln Gln Ala Gly 210 215 220Tyr Pro Phe Thr Asp Asp Met Asn Gly Phe Gln Gln Glu Gly Phe Gly225 230 235 240Trp Met Asp Met Thr Ile Tyr Glu Asp Phe Pro Arg Ala Val Tyr His 245 250 255Thr Glu Ile Ser Tyr Ile Ile Cys Ile Gly Tyr Cys Tyr Asn Lys Asn 260 265 270Val Tyr Phe Val Gly Lys Arg Trp Asn Thr Ala Ser Ala Tyr Leu Arg 275 280 285Pro Ala Leu Ser Arg Pro Asn Leu Ser Ala Glu Val Ser Thr Leu Val 290 295 300Thr Lys Val Leu Phe Glu Gly Thr Lys Ala Ile Gly Ile Glu Tyr Ile305 310 315 320Lys Asn Gly Glu Lys Lys Lys Val Phe Ala Ser Lys Glu Val Ile Leu 325 330 335Ser Gly Gly Ala Ile Asn Ser Pro Gln Leu Leu Met Leu Ser Gly Val 340 345 350Gly Val Gly Gln Asn Leu Gln Asp His Leu Glu Val Tyr Ile Gln Gln 355 360 365Lys Cys Thr Gln Pro Leu Thr Leu Tyr Lys Ser Gln Lys Pro Leu Gln 370 375 380Met Ile Lys Ile Gly Leu Glu Trp Phe Trp Lys Ser Thr Gly Asp Gly385 390 395 400Ala Thr Ala His Leu Glu Thr Gly Gly Phe Ile Arg Ser Arg Pro Gly 405 410 415Ile Ser His Pro Asp Ile Gln Phe His Phe Leu Pro Ser Gln Val Ile 420 425 430Asp His Gly Arg Val Ala Ser Gln Leu Glu Ala Tyr Gln Val His Ile 435 440 445Gly Pro Met Arg Ser Thr Ser Val Gly Lys Leu Lys Leu Lys Ser Ser 450 455 460Asp Pro Thr Glu His Pro Ile Leu Glu Pro Asn Tyr Leu Ser Thr Glu465 470 475 480Met Asp Val Trp Glu Phe Arg Gln Cys Val Lys Leu Ala Arg Glu Ile 485 490 495Phe Ala Gln Lys Ala Phe Glu Glu Phe Arg Gly Pro Glu Ile Gln Pro 500 505 510Gly Glu His Ile Gln Ser Asp Lys Glu Ile Asp Ala Phe Ile Arg Gln 515 520 525Lys Ser Asp Ser Ala Tyr His Pro Ser Cys Thr Cys Lys Met Gly Gln 530 535 540Asn Ser Asp Pro Met Ala Val Val Asn Pro Glu Thr Lys Val Ile Gly545 550 555 560Thr Glu Asn Leu Arg Val Val Asp Ala Ser Ile Met Pro Ser Ile Val 565 570 575Ser Gly Asn Leu Asn Ala Pro Thr Ile Met Ile Ala Glu Lys Ala Ala

580 585 590Asp Ile Ile Leu Gly Leu Pro Pro Leu Gln Glu Lys Asn Val Pro Val 595 600 605Tyr Lys Pro Lys Thr Leu Glu Thr Gln His Asn Pro Asp Lys Val Ala 610 615 620Trp Gln Asn Lys Ser Ile Tyr Pro Met Leu Ile Lys625 630 63515140PRTHuman 15Leu Leu Tyr Lys Pro Val Asp Arg Val Thr Arg Ser Thr Leu Val Leu1 5 10 15His Asp Leu Leu Lys His Thr Pro Ala Ser His Pro Asp His Pro Leu 20 25 30Leu Gln Asp Ala Leu Arg Ile Ser Gln Asn Phe Leu Ser Ser Ile Asn 35 40 45Glu Glu Ile Thr Pro Arg Arg Gln Ser Met Thr Val Lys Lys Gly Glu 50 55 60Gly Glu Asp Arg Met Lys Ala Ser Ser Thr Arg Lys Arg Leu Leu Leu65 70 75 80Met Glu Glu Ala Leu Gln Arg Pro Val Ala Ser Asp Phe Glu Pro Gln 85 90 95Gly Leu Ser Glu Ala Ala Arg Trp Asn Ser Lys Glu Asn Leu Leu Ala 100 105 110Gly Pro Ser Glu Asn Asp Pro Asn Leu Phe Val Ala Leu Tyr Asp Phe 115 120 125Val Ala Ser Gly Asp Asn Thr Leu Ser Ile Thr Lys 130 135 14016109PRTHuman 16Met Leu Met Ala Gln Glu Ala Leu Ala Phe Leu Met Ala Gln Gly Ala1 5 10 15Met Leu Ala Ala Gln Glu Arg Arg Val Pro Arg Ala Ala Glu Val Pro 20 25 30Gly Ala Gln Gly Gln Gln Gly Pro Arg Gly Arg Glu Glu Ala Pro Arg 35 40 45Gly Val Arg Met Ala Val Pro Leu Leu Arg Arg Met Glu Gly Ala Pro 50 55 60Ala Gly Pro Gly Gly Arg Thr Ala Ala Cys Phe Ser Cys Thr Ser Arg65 70 75 80Cys Leu Ser Arg Arg Pro Trp Lys Arg Ser Trp Ser Ala Gly Ser Cys 85 90 95Pro Gly Met Pro His Leu Ser Pro Asp Gln Gly Arg Phe 100 10517222PRTHuman 17Met Phe Ser Leu Ser Thr Val Ala Phe Leu Val Leu Leu Ser Thr Ser1 5 10 15Gly His Ala Ser Met Cys Pro Asp Asn Asn Gly Met Ser Asp Glu Val 20 25 30Arg Asn Thr Phe Leu Lys Lys His Asn Ala Tyr Arg Thr Leu Val Ala 35 40 45Lys Gly Glu Ala Lys Asn Ala Lys Glu Ile Gly Gly Tyr Ala Pro Lys 50 55 60Ala Ala Arg Met Leu Lys Val Thr Tyr Asp Cys Ala Ile Glu Glu Asn65 70 75 80Thr Met Asn Phe Ala Lys Lys Cys Val Phe Ala His Asn Ser Tyr Lys 85 90 95Asp Arg Asn Tyr Trp Gly Gln Asn Phe Tyr Met Thr Ser Ile Leu Asn 100 105 110Gln Asn Lys Thr Val Ala Ala Ala Glu Ser Val Asp Leu Trp Phe Asp 115 120 125Glu Leu Gln Gln Asn Gly Val Pro Val Asp Asn Val Met Thr Met Ala 130 135 140Val Phe Asn Arg Gly Val Gly His Tyr Thr Gln Val Val Trp Gln Trp145 150 155 160Ser Asn Lys Ile Gly Cys Ala Val Glu Trp Cys Ser Asp Met Thr Phe 165 170 175Val Ala Cys Glu Tyr Asp Ser Ala Gly Asn Tyr Met Gly Met Pro Ile 180 185 190Tyr Glu Val Gly Asn Pro Cys Thr Asn Asn Glu Asp Cys Lys Cys Thr 195 200 205Asn Cys Val Cys Ser Arg Glu Glu Ala Leu Cys Ile Ala Pro 210 215 22018702PRTHuman 18Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln1 5 10 15Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr 20 25 30Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly 35 40 45Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly 50 55 60Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile65 70 75 80Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser 85 90 95Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile 100 105 110Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp 115 120 125Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu 130 135 140Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys145 150 155 160Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr 165 170 175Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln 180 185 190Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn 195 200 205Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg 210 215 220Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro225 230 235 240Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn 245 250 255Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe 260 265 270Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn 275 280 285Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser 290 295 300Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala305 310 315 320Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu 325 330 335Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr 340 345 350Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg 355 360 365Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr 370 375 380Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Glu Leu Ser385 390 395 400Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp 405 410 415Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn 420 425 430Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser 435 440 445Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile 450 455 460Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn465 470 475 480Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val 485 490 495Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro 500 505 510Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln 515 520 525Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser 530 535 540Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn545 550 555 560Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser 565 570 575Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly 580 585 590Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly 595 600 605Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln 610 615 620Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu625 630 635 640Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe 645 650 655Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile 660 665 670Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr 675 680 685Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile 690 695 70019278PRTHuman 19Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser1 5 10 15Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln 20 25 30Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Met Leu Phe Gln Arg Leu 35 40 45Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu 50 55 60Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Leu Ile Thr Tyr Leu Asn65 70 75 80Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gln Thr Pro Gly Arg Ala 85 90 95Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val 100 105 110Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile 115 120 125Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile 130 135 140Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu145 150 155 160Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile Ile Asn 165 170 175Asp Tyr Glu Glu Phe Ser Lys Gly Glu Glu Leu Cys Gly Val Met Thr 180 185 190Ile Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Ser Gln Thr Leu Asp 195 200 205Lys Val Tyr Gln Met Lys Ser Lys Pro Arg Gly Tyr Cys Leu Ile Ile 210 215 220Asn Asn His Asn Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Leu His225 230 235 240Ser Ile Arg Asp Arg Asn Gly Thr His Leu Asp Ala Gly Ser His Ser 245 250 255Val Ala Gln Ala Gly Val Gln Trp Cys Asp Leu Gly Ser Leu Gln Pro 260 265 270Pro Pro Pro Trp Phe Gly 27520303PRTHuman 20Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala1 5 10 15Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly 35 40 45Leu Pro Ile Asn Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr65 70 75 80Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val 85 90 95Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110Pro Ala Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly145 150 155 160Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg225 230 235 240Asp Val Ser Leu Pro Arg Gly Ala Phe Pro Pro Arg Gly Pro Arg Pro 245 250 255Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285Ala Leu Gln His Ser Tyr Leu His Lys Asp Glu Gly Asn Pro Glu 290 295 30021823PRTHuman 21Met Thr Val Leu Gln Glu Pro Val Gln Ala Ala Ile Trp Gln Ala Leu1 5 10 15Asn His Tyr Ala Tyr Arg Asp Ala Val Phe Leu Ala Glu Arg Leu Tyr 20 25 30Ala Glu Val His Ser Glu Glu Ala Leu Phe Leu Leu Ala Thr Cys Tyr 35 40 45Tyr Arg Ser Gly Lys Ala Tyr Lys Ala Tyr Arg Leu Leu Lys Gly His 50 55 60Ser Cys Thr Thr Pro Gln Cys Lys Tyr Leu Leu Ala Lys Cys Cys Val65 70 75 80Asp Leu Ser Lys Leu Ala Glu Gly Glu Gln Ile Leu Ser Gly Gly Val 85 90 95Phe Asn Lys Gln Lys Ser His Asp Asp Ile Val Thr Glu Phe Gly Asp 100 105 110Ser Ala Cys Phe Thr Leu Ser Leu Leu Gly His Val Tyr Cys Lys Thr 115 120 125Asp Arg Leu Ala Lys Gly Ser Glu Cys Tyr Gln Lys Ser Leu Ser Leu 130 135 140Asn Pro Phe Leu Trp Ser Pro Phe Glu Ser Leu Cys Glu Ile Gly Glu145 150 155 160Lys Pro Asp Pro Asp Gln Thr Phe Lys Phe Thr Ser Leu Gln Asn Phe 165 170 175Ser Asn Cys Leu Pro Asn Ser Cys Thr Thr Gln Val Pro Asn His Ser 180 185 190Leu Ser His Arg Gln Pro Glu Thr Val Leu Thr Glu Thr Pro Gln Asp 195 200 205Thr Ile Glu Leu Asn Arg Leu Asn Leu Glu Ser Ser Asn Ser Lys Tyr 210 215 220Ser Leu Asn Thr Asp Ser Ser Val Ser Tyr Ile Asp Ser Ala Val Ile225 230 235 240Ser Pro Asp Thr Val Pro Leu Gly Thr Gly Thr Ser Ile Leu Ser Lys 245 250 255Gln Val Gln Asn Lys Pro Lys Thr Gly Arg Ser Leu Leu Gly Gly Pro 260 265 270Ala Ala Leu Ser Pro Leu Thr Pro Ser Phe Gly Ile Leu Pro Leu Glu 275 280 285Thr Pro Ser Pro Gly Asp Gly Ser Tyr Leu Gln Asn Tyr Thr Asn Thr 290 295 300Pro Pro Val Ile Asp Val Pro Ser Thr Gly Ala Pro Ser Lys Lys Ser305 310 315 320Val Ala Arg Ile Gly Gln Thr Gly Thr Lys Ser Val Phe Ser Gln Ser 325 330 335Gly Asn Ser Arg Glu Val Thr Pro Ile Leu Ala Gln Thr Gln Ser Ser 340 345 350Gly Pro Gln Thr Ser Thr Thr Pro Gln Val Leu Ser Pro Thr Ile Thr 355 360 365Ser Pro Pro Asn Ala Leu Pro Arg Arg Ser Ser Arg Leu Phe Thr Ser 370 375 380Asp Ser Ser Thr Thr Lys Glu Asn Ser Lys Lys Leu Lys Met Lys Phe385 390 395 400Pro Pro Lys Ile Pro Asn Arg Lys Thr Lys Ser Lys Thr Asn Lys Gly 405 410 415Gly Ile Thr Gln Pro Asn Ile Asn Asp Ser Leu Glu Ile Thr Lys Leu 420 425 430Asp Ser Ser Ile Ile Ser Glu Gly Lys Ile Ser Thr Ile Thr Pro Gln 435 440 445Ile Gln Ala Phe Asn Leu Gln Lys Ala Ala Ala Gly Leu Met Ser Leu 450 455 460Leu Arg Glu Met Gly Lys Gly Tyr Leu Ala Leu Cys Ser Tyr Asn Cys465 470 475 480Lys Glu Ala Ile Asn Ile Leu Ser His Leu Pro Ser His His Tyr Asn 485 490 495Thr Gly Trp Val Leu Cys Gln Ile Gly Arg Ala Tyr Phe Glu Leu Ser 500 505 510Glu Tyr Met Gln Ala Glu Arg Ile Phe Ser Glu Val Arg Arg Ile Glu 515 520 525Asn Tyr Arg Val Glu Gly Met Glu Ile Tyr Ser Thr Thr Leu Trp His 530 535 540Leu Gln Lys Asp Val Ala Leu Ser Val Leu Ser Lys Asp Leu Thr Asp545 550 555 560Met Asp Lys Asn Ser Pro Glu Ala Trp Cys Ala Ala Gly Asn Cys Phe 565 570 575Ser Leu Gln Arg Glu His Asp Ile Ala Ile Lys Phe Phe Gln Arg Ala 580 585 590Ile Gln Val Asp Pro Asn Tyr Ala Tyr Ala Tyr Thr Leu Leu Gly His 595 600 605Glu Phe Val Leu Thr Glu Glu Leu Asp Lys Ala Leu Ala Cys Phe Arg 610 615 620Asn Ala Ile Arg Val Asn Pro Arg His Tyr Asn Ala Trp Tyr Gly Leu625 630 635 640Gly Met Ile Tyr Tyr Lys Gln Glu Lys Phe Ser Leu Ala Glu Met His 645 650 655Phe Gln Lys Ala Leu Asp Ile Asn Pro Gln Ser Ser

Val Leu Leu Cys 660 665 670His Ile Gly Val Val Gln His Ala Leu Lys Lys Ser Glu Lys Ala Leu 675 680 685Asp Thr Leu Asn Lys Ala Ile Val Ile Asp Pro Lys Asn Pro Leu Cys 690 695 700Lys Phe His Arg Ala Ser Val Leu Phe Arg Asn Glu Lys Tyr Lys Ser705 710 715 720Ala Leu Gln Glu Leu Glu Glu Leu Lys Gln Ile Val Pro Lys Glu Ser 725 730 735Leu Val Tyr Phe Leu Ile Gly Lys Val Tyr Lys Lys Leu Gly Gln Thr 740 745 750His Leu Ala Leu Met Asn Phe Ser Trp Ala Met Asp Leu Asp Pro Lys 755 760 765Gly Ala Asn Asn Gln Ile Lys Glu Ala Ile Asp Lys Arg Tyr Leu Pro 770 775 780Asp Asp Glu Glu Pro Ile Thr Gln Glu Glu Gln Ile Met Gly Thr Asp785 790 795 800Glu Ser Gln Glu Ser Ser Met Thr Asp Ala Asp Asp Thr Gln Leu His 805 810 815Ala Ala Glu Ser Asp Glu Phe 82022382PRTHuman 22Met Gly Leu Leu Leu Pro Leu Ala Leu Cys Ile Leu Val Leu Cys Cys1 5 10 15Gly Ala Met Ser Pro Pro Gln Leu Ala Leu Asn Pro Ser Ala Leu Leu 20 25 30Ser Arg Gly Cys Asn Asp Ser Asp Val Leu Ala Val Ala Gly Phe Ala 35 40 45Leu Arg Asp Ile Asn Lys Asp Arg Lys Asp Gly Tyr Val Leu Arg Leu 50 55 60Asn Arg Val Asn Asp Ala Gln Glu Tyr Arg Arg Gly Gly Leu Gly Ser65 70 75 80Leu Phe Tyr Leu Thr Leu Asp Val Leu Glu Thr Asp Cys His Val Leu 85 90 95Arg Lys Lys Ala Trp Gln Asp Cys Gly Met Arg Ile Phe Phe Glu Ser 100 105 110Val Tyr Gly Gln Cys Lys Ala Ile Phe Tyr Met Asn Asn Pro Ser Arg 115 120 125Val Leu Tyr Leu Ala Ala Tyr Asn Cys Thr Leu Arg Pro Val Ser Lys 130 135 140Lys Lys Ile Tyr Met Thr Cys Pro Asp Cys Pro Ser Ser Ile Pro Thr145 150 155 160Asp Ser Ser Asn His Gln Val Leu Glu Ala Ala Thr Glu Ser Leu Ala 165 170 175Lys Tyr Asn Asn Glu Asn Thr Ser Lys Gln Tyr Ser Leu Phe Lys Val 180 185 190Thr Arg Ala Ser Ser Gln Trp Val Val Gly Pro Ser Tyr Phe Val Glu 195 200 205Tyr Leu Ile Lys Glu Ser Pro Cys Thr Lys Ser Gln Ala Ser Ser Cys 210 215 220Ser Leu Gln Ser Ser Asp Ser Val Pro Val Gly Leu Cys Lys Gly Ser225 230 235 240Leu Thr Arg Thr His Trp Glu Lys Phe Val Ser Val Thr Cys Asp Phe 245 250 255Phe Glu Ser Gln Ala Pro Ala Thr Gly Ser Glu Asn Ser Ala Val Asn 260 265 270Gln Lys Pro Thr Asn Leu Pro Lys Val Glu Glu Ser Gln Gln Lys Asn 275 280 285Thr Pro Pro Thr Asp Ser Pro Ser Lys Ala Gly Pro Arg Gly Pro Val 290 295 300Gln Tyr Leu Pro Asp Leu Asp Asp Lys Asn Ser Gln Glu Lys Gly Pro305 310 315 320Gln Glu Ala Phe Pro Val His Leu Asp Leu Thr Thr Asn Pro Gln Gly 325 330 335Glu Thr Leu Asp Ile Ser Phe Leu Phe Leu Glu Pro Met Glu Glu Lys 340 345 350Leu Val Val Leu Pro Phe Pro Lys Glu Lys Ala Arg Thr Ala Glu Cys 355 360 365Pro Gly Pro Ala Gln Asn Ala Ser Pro Leu Val Leu Pro Pro 370 375 38023257PRTHuman 23Met Pro Phe Ile Ser His His Tyr Pro His Asp His Ser Cys Arg Trp1 5 10 15Val Glu Pro Phe Ile Gly Gly Gly Ala Val Phe Leu Asn Met Phe Ala 20 25 30Gln Asn Ala Leu Leu Ala Asp Ser Asn Pro Asp Leu Ile Asn Leu Tyr 35 40 45Arg Thr Ile Gln Arg Gln Lys Thr Asn Phe Ile Asn Gln Val Gln Asn 50 55 60Leu Ala Asp Lys Thr Phe Val Glu Lys Asp Tyr Tyr Glu Met Arg Asp65 70 75 80Arg Phe Asn Lys Thr Cys Ile Ser Gly Gln Pro Leu Gln Arg Ala Ala 85 90 95Leu Phe Tyr Ser Leu Asn Arg Leu Gly Tyr Asn Gly Met Cys Arg Tyr 100 105 110Asn Ser Glu Arg Ile Tyr Ser Val Pro Trp Gly Lys His Thr Glu Leu 115 120 125Lys Leu Asp Phe Asn Lys Ile Asp Tyr Leu Ser Phe Arg Leu Ser Gly 130 135 140Ile Glu Leu Ile Thr Ala Gly Phe Glu Glu Thr Leu Ala Ala Thr Gly145 150 155 160Glu Gly Asp Gln Ile Tyr Cys Asp Pro Pro Tyr Asp Lys Thr Ser Lys 165 170 175Thr Ser Phe Val Ser Tyr Asp Gly Lys Pro Phe Ser Gln Ser Asp His 180 185 190Val Leu Leu Ala Asn Met Leu Val Asp Ala His Arg Lys Gly Ala Ala 195 200 205Val Ala Ile Ser Asn Ser Leu Thr Pro Phe Thr Leu Gly Leu Tyr Glu 210 215 220Glu Arg Gly Phe Val Ile His Arg Leu Ser Ala Tyr Arg Ser Val Gly225 230 235 240Ser Lys Pro Asn Thr Arg Lys Thr Glu Thr Glu Ile Leu Ala Val Leu 245 250 255Lys24288PRTHuman 24Met Asn Ala Lys Lys Asn Pro Leu Val Lys Pro Phe Leu Lys Trp Ala1 5 10 15Gly Gly Lys Arg Gln Leu Leu Pro Glu Ile Leu Lys Tyr Leu Pro Lys 20 25 30Asn Ile Gly Lys Thr Thr Tyr Phe Glu Pro Phe Leu Gly Gly Gly Ala 35 40 45Leu Leu Phe Glu Leu Gln Pro Lys Gln Ala Ile Val Asn Asp Ser Asn 50 55 60Arg Glu Leu Ile Asn Cys Tyr Arg Val Ile Lys Asp Asn Val Glu Glu65 70 75 80Leu Ile Glu Val Leu Lys Val His Lys Ala Lys Asn Ser Lys Glu Tyr 85 90 95Phe Asp Tyr Leu Arg Glu Arg Asp Arg Leu Lys Gln Tyr Asn Lys Phe 100 105 110Ser Asp Ile Gln Lys Ala Ala Arg Ile Ile Tyr Leu Asn Lys Thr Cys 115 120 125Tyr Asn Gly Leu Phe Arg Val Asn Ser Lys Gly Gln Phe Asn Val Pro 130 135 140Phe Gly Ser Tyr Lys Asn Pro Asn Ile Leu Asp Glu Ala Val Leu Arg145 150 155 160Gly Val Asn Asp Tyr Leu Asn Gln Lys Ser Val Thr Phe Leu Asn Ile 165 170 175Asp Phe Ala Glu Ala Val Lys Asp Ala Lys Lys Gly Asp Phe Val Tyr 180 185 190Phe Asp Pro Pro Tyr Asp Pro Val Ser Asn Thr Ala Ser Phe Thr Gly 195 200 205Tyr Asp Ile Asn Gly Phe Asn Gln Asn Glu Gln Arg Arg Leu Lys Gln 210 215 220Val Val Asp Glu Leu Thr Glu Lys Gly Cys Asn Val Met Leu Ser Asn225 230 235 240Ser Ala Thr Asp Phe Ile Leu Asp Leu Tyr Lys Asp His Lys Tyr Thr 245 250 255Ile Glu Thr Val Ser Ala Thr Arg Ser Ile Asn Ser Asn Ala Leu Lys 260 265 270Arg Gly Lys Ile Asn Glu Val Leu Val Leu Asn Tyr Val Pro Lys Leu 275 280 28525395PRTHuman 25Gly Pro Gly Cys Asn Thr Lys Lys Pro Asn Leu Asp Ala Glu Leu Asp1 5 10 15Gln Leu Leu Gln Gly His Tyr Ile Lys Gly Tyr Pro Lys Gln Tyr Thr 20 25 30Tyr Phe Leu Glu Asp Gly Lys Val Lys Val Ser Arg Pro Glu Gly Val 35 40 45Lys Ile Ile Pro Pro Gln Ser Asp Arg Gln Lys Ile Val Leu Gln Ala 50 55 60His Asn Leu Ala His Thr Gly Arg Glu Ala Thr Leu Leu Lys Ile Ala65 70 75 80Asn Leu Tyr Trp Trp Pro Asn Met Arg Lys Asp Val Val Lys Gln Leu 85 90 95Gly Arg Cys Gln Gln Cys Leu Ile Thr Asn Ala Ser Asn Lys Ala Ser 100 105 110Gly Pro Ile Leu Arg Pro Asp Arg Pro Gln Lys Pro Phe Asp Lys Phe 115 120 125Phe Ile Asp Tyr Ile Gly Pro Leu Pro Pro Ser Gln Gly Tyr Leu Tyr 130 135 140Val Leu Val Val Val Asp Gly Met Thr Gly Phe Thr Trp Leu Tyr Pro145 150 155 160Thr Lys Ala Pro Ser Thr Ser Ala Thr Val Lys Ser Leu Asn Val Leu 165 170 175Thr Ser Ile Ala Ile Pro Lys Val Ile His Ser Asp Gln Gly Ala Ala 180 185 190Phe Thr Ser Ser Thr Phe Ala Glu Trp Ala Lys Glu Arg Gly Ile His 195 200 205Leu Glu Phe Ser Thr Pro Tyr His Pro Gln Ser Ser Gly Lys Val Glu 210 215 220Arg Lys Asn Ser Asp Ile Lys Arg Leu Leu Thr Lys Leu Leu Val Gly225 230 235 240Arg Pro Thr Lys Trp Tyr Asp Leu Leu Pro Val Val Gln Leu Ala Leu 245 250 255Asn Asn Thr Tyr Ser Pro Val Leu Lys Tyr Thr Pro His Gln Leu Leu 260 265 270Phe Gly Ile Asp Ser Asn Thr Pro Phe Ala Asn Gln Asp Thr Leu Asp 275 280 285Leu Thr Arg Glu Glu Glu Leu Ser Leu Leu Gln Glu Ile Arg Thr Ser 290 295 300Leu Tyr His Pro Ser Thr Pro Pro Ala Ser Ser Arg Ser Trp Ser Pro305 310 315 320Val Val Gly Gln Leu Val Gln Glu Arg Val Ala Arg Pro Ala Ser Leu 325 330 335Arg Pro Arg Trp His Lys Pro Ser Thr Val Leu Lys Val Leu Asn Pro 340 345 350Arg Thr Val Val Ile Leu Asp His Leu Gly Asn Asn Arg Thr Val Ser 355 360 365Ile Asp Asn Leu Lys Pro Thr Ser His Gln Asn Gly Thr Thr Asn Asp 370 375 380Thr Ala Thr Met Asp His Leu Glu Lys Asn Glu385 390 39526395PRTHuman 26Gly Pro Gly Cys Asn Thr Lys Lys Pro Asn Leu Asp Ala Glu Leu Asp1 5 10 15Gln Leu Leu Gln Gly His Tyr Ile Lys Gly Tyr Pro Lys Gln Tyr Thr 20 25 30Tyr Phe Leu Glu Asp Gly Lys Val Lys Val Ser Arg Pro Glu Gly Val 35 40 45Lys Ile Ile Pro Pro Gln Ser Asp Arg Gln Lys Ile Val Leu Gln Ala 50 55 60His Asn Leu Ala His Thr Gly Arg Glu Ala Thr Leu Leu Lys Ile Ala65 70 75 80Asn Leu Tyr Trp Trp Pro Asn Met Arg Lys Asp Val Val Lys Gln Leu 85 90 95Gly Arg Cys Gln Gln Cys Leu Ile Thr Asn Ala Ser Asn Lys Ala Ser 100 105 110Gly Pro Ile Leu Arg Pro Asp Arg Pro Gln Lys Pro Phe Asp Lys Phe 115 120 125Phe Ile Asp Tyr Ile Gly Pro Leu Pro Pro Ser Gln Gly Tyr Leu Tyr 130 135 140Val Leu Val Val Val Asp Gly Met Thr Gly Phe Thr Trp Leu Tyr Pro145 150 155 160Thr Lys Ala Pro Ser Thr Ser Ala Thr Val Lys Ser Leu Asn Val Leu 165 170 175Thr Ser Ile Ala Ile Pro Lys Val Ile His Ser Asp Gln Gly Ala Ala 180 185 190Phe Thr Ser Ser Thr Phe Ala Glu Trp Ala Lys Glu Arg Gly Ile His 195 200 205Leu Glu Phe Ser Thr Pro Tyr His Pro Gln Ser Ser Gly Lys Val Glu 210 215 220Arg Lys Asn Ser Asp Ile Lys Arg Leu Leu Thr Lys Leu Leu Val Gly225 230 235 240Arg Pro Thr Lys Trp Tyr Asp Leu Leu Pro Val Val Gln Leu Ala Leu 245 250 255Asn Asn Thr Tyr Ser Pro Val Leu Lys Tyr Thr Pro His Gln Leu Leu 260 265 270Phe Gly Ile Asp Ser Asn Thr Pro Phe Ala Asn Gln Asp Thr Leu Asp 275 280 285Leu Thr Arg Glu Glu Glu Leu Ser Leu Leu Gln Glu Ile Arg Thr Ser 290 295 300Leu Tyr His Pro Ser Thr Pro Pro Ala Ser Ser Arg Ser Trp Ser Pro305 310 315 320Val Val Gly Gln Leu Val Gln Glu Arg Val Ala Arg Pro Ala Ser Leu 325 330 335Arg Pro Arg Trp His Lys Pro Ser Thr Val Leu Lys Val Leu Asn Pro 340 345 350Arg Thr Val Val Ile Leu Asp His Leu Gly Asn Asn Arg Thr Val Ser 355 360 365Ile Asp Asn Leu Lys Pro Thr Ser His Gln Asn Gly Thr Thr Asn Asp 370 375 380Thr Ala Thr Met Asp His Leu Glu Lys Asn Glu385 390 39527407PRTHuman 27Met Gly Val Leu Thr Lys Ser Leu Gln Lys Glu Ser Ala Lys Thr Asn1 5 10 15Lys Arg Asp Asp Tyr Ser Ala Ala Asp Leu Met His Val Pro Thr Gly 20 25 30Phe Asp Ala Ile Asp Tyr Glu Gly Gly Thr Ile Val Glu Asp Ile Asp 35 40 45Gly Asp Pro Met Leu Asn Ile Gly Leu Pro Met Gly Lys Ile Ile Leu 50 55 60Cys Cys Gly Asn Ser Gln Ala Gly Lys Thr Thr Gly Ala Leu Gln Phe65 70 75 80Ala Asn Gly Met Ala Ser His Leu Asp Gly Asp Val Val Ile Phe Asp 85 90 95Phe Glu Arg Gly Ile Leu Asp Pro Arg Ser Arg Ile Arg Asn Leu Cys 100 105 110Arg Leu Ser Asn Asp Glu Tyr Asp Asn Arg Phe Thr Ile Tyr Lys Asn 115 120 125Ala Gly Met Ser Val Glu Phe Phe Lys Lys Gln Ile Phe Lys Ile Val 130 135 140Glu Leu Lys Glu Lys Leu Ala Lys Ala Asp Met Val Asp Trp Tyr Met145 150 155 160Leu Asn Gly Ala Pro Val Lys Ile Tyr Pro Pro Thr Tyr Val Leu Leu 165 170 175Asp Ser Ile Pro Ser Met Lys Pro Glu Asp Val Leu Asn Asp Ser Ser 180 185 190Leu Asp Asn Asn Met Val Phe Ser Lys Met Ala Ala Ala Asn Ser Ala 195 200 205Met Leu Thr Ser Ile Val Asn Val Leu Glu Lys Tyr Asn Ile Thr Leu 210 215 220Ile Cys Ile Asn His Ile Thr Thr Lys Ile Ile Ile Asn Ala Tyr Gly225 230 235 240Pro Arg Lys Val Leu Leu Pro Gly Met Glu Pro Glu Glu Asn Leu Pro 245 250 255Gly Gly Asn Lys Phe Val Tyr Leu Pro Ser Tyr Val Leu Lys Phe Ala 260 265 270Ser Gly Lys Ala Leu Asn Lys Asp Lys Asp Phe Lys Val Asn Gly Arg 275 280 285Val Thr Asn Cys Thr Phe Leu Lys Ser Arg Ser Ser Phe Asn Gly Ala 290 295 300Lys Leu Pro Leu Ala Val Thr Glu Lys His Gly Phe Ser Asn Val Met305 310 315 320Thr Asn Ile Leu Ala Met Lys Glu Glu Lys Met Leu Lys Gly Thr Gly 325 330 335Gln Gly Gly Phe Trp Phe Glu Gly His Glu Asp Met Lys Phe Lys Gln 340 345 350Ser Glu Phe Ile Lys Lys Tyr Asn Lys Asp Thr Glu Phe Gln Glu Met 355 360 365Phe Asp Glu Val Ser Ser Glu Phe Trp Gln Gly Arg Leu Glu Asp Arg 370 375 380Phe Gly Asp Glu Tyr Asp Ser Val Glu Lys Ser Lys Gly Asn Asp Phe385 390 395 400Asp Asp Asp Asp Asp Asp Glu 40528661PRTHuman 28Met Asp Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val Ile Gly1 5 10 15Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pro Arg Asn Gln Asp Trp 20 25 30Leu Gly Val Ser Arg Gln Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu 35 40 45Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys Trp Arg Gly Gly 50 55 60Gln Val Ser Leu Lys Val Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala65 70 75 80Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro Gly Ser Gln Lys Val 85 90 95Leu Pro Asp Gly Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn Gly 100 105 110Ser Gln Val Trp Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp 115 120 125Ala Cys Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser 130 135 140Gln Lys Arg Ser Phe Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp145 150 155 160Gln Val Leu Gly Gly Pro Val Ser Gly Leu Ser Ile Gly Thr Gly Arg 165 170 175Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr His Arg Arg 180

185 190Gly Ser Arg Ser Tyr Val Pro Leu Ala His Ser Ser Ser Ala Phe Thr 195 200 205Ile Thr Asp Gln Val Pro Phe Ser Val Ser Val Ser Gln Leu Arg Ala 210 215 220Leu Asp Gly Gly Asn Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe225 230 235 240Ala Leu Gln Leu His Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu 245 250 255Ser Tyr Thr Trp Asp Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg 260 265 270Ala Leu Val Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala 275 280 285Gln Val Val Leu Gln Ala Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser 290 295 300Pro Val Pro Gly Thr Thr Asp Gly His Arg Pro Thr Ala Glu Ala Pro305 310 315 320Asn Thr Thr Ala Gly Gln Val Pro Thr Thr Glu Val Val Gly Thr Thr 325 330 335Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr Ser Val Gln 340 345 350Val Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr 355 360 365Ala Glu Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser Glu Val 370 375 380Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly Met385 390 395 400Thr Pro Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 405 410 415Gln Val Thr Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro 420 425 430Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser Thr Glu 435 440 445Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu 450 455 460Arg Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr465 470 475 480Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala 485 490 495Glu Ile Leu Gln Ala Val Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu 500 505 510Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys Met Glu Ile 515 520 525Ser Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val 530 535 540Leu Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln Ile Leu Lys Gly545 550 555 560Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser 565 570 575Leu Ala Val Val Ser Thr Gln Leu Ile Met Pro Gly Gln Glu Ala Gly 580 585 590Leu Gly Gln Val Pro Leu Ile Val Gly Ile Leu Leu Val Leu Met Ala 595 600 605Val Val Leu Ala Ser Leu Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp 610 615 620Phe Ser Val Pro Gln Leu Pro His Ser Ser Ser His Trp Leu Arg Leu625 630 635 640Pro Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu 645 650 655Ser Gly Gln Gln Val 66029648PRTHuman 29Met Ser His His Gly Gly Ala Pro Lys Ala Ser Thr Trp Val Val Ala1 5 10 15Ser Arg Arg Ser Ser Thr Val Ser Arg Ala Pro Glu Arg Arg Pro Ala 20 25 30Glu Glu Leu Asn Arg Thr Gly Pro Glu Gly Tyr Ser Val Gly Arg Gly 35 40 45Gly Arg Trp Arg Gly Thr Ser Arg Pro Pro Glu Ala Val Ala Ala Gly 50 55 60His Glu Glu Leu Pro Leu Cys Phe Ala Leu Lys Ser His Phe Val Gly65 70 75 80Ala Val Ile Gly Arg Gly Gly Ser Lys Ile Lys Asn Ile Gln Ser Thr 85 90 95Thr Asn Thr Thr Ile Gln Ile Ile Gln Glu Gln Pro Glu Ser Leu Val 100 105 110Lys Ile Phe Gly Ser Lys Ala Met Gln Thr Lys Ala Lys Ala Val Ile 115 120 125Asp Asn Phe Val Lys Lys Leu Glu Glu Asn Tyr Asn Ser Glu Cys Gly 130 135 140Ile Asp Thr Ala Phe Gln Pro Ser Val Gly Lys Asp Gly Ser Thr Asp145 150 155 160Asn Asn Val Val Ala Gly Asp Arg Pro Leu Ile Asp Trp Asp Gln Ile 165 170 175Arg Glu Glu Gly Leu Lys Trp Gln Lys Thr Lys Trp Ala Asp Leu Pro 180 185 190Pro Ile Lys Lys Asn Phe Tyr Lys Glu Ser Thr Ala Thr Ser Ala Met 195 200 205Ser Lys Val Glu Ala Asp Ser Trp Arg Lys Glu Asn Phe Asn Ile Thr 210 215 220Trp Asp Asp Leu Lys Asp Gly Glu Lys Arg Pro Ile Pro Asn Pro Thr225 230 235 240Cys Thr Phe Asp Asp Ala Phe Gln Cys Tyr Pro Glu Val Met Glu Asn 245 250 255Ile Lys Lys Ala Gly Phe Gln Lys Pro Thr Pro Ile Gln Ser Gln Ala 260 265 270Trp Pro Ile Val Leu Gln Gly Ile Asp Leu Ile Gly Val Ala Gln Thr 275 280 285Gly Thr Gly Lys Thr Leu Cys Tyr Leu Met Pro Gly Phe Ile His Leu 290 295 300Val Leu Gln Pro Ser Leu Lys Gly Gln Arg Asn Arg Pro Gly Met Leu305 310 315 320Val Leu Thr Pro Thr Arg Glu Leu Ala Leu Gln Val Glu Gly Glu Cys 325 330 335Cys Lys Tyr Ser Tyr Lys Gly Leu Arg Ser Val Cys Val Tyr Gly Gly 340 345 350Gly Asn Arg Asp Glu Gln Ile Glu Glu Leu Lys Lys Gly Val Asp Ile 355 360 365Ile Ile Ala Thr Pro Gly Arg Leu Asn Asp Leu Gln Met Ser Asn Phe 370 375 380Val Asn Leu Lys Asn Ile Thr Tyr Leu Val Leu Asp Glu Ala Asp Lys385 390 395 400Met Leu Asp Met Gly Phe Glu Pro Gln Ile Met Lys Ile Leu Leu Asp 405 410 415Val Arg Pro Asp Arg Gln Thr Val Met Thr Ser Ala Thr Trp Pro His 420 425 430Ser Val His Arg Leu Ala Gln Ser Tyr Leu Lys Glu Pro Met Ile Val 435 440 445Tyr Val Gly Thr Leu Asp Leu Val Ala Val Ser Ser Val Lys Gln Asn 450 455 460Ile Ile Val Thr Thr Glu Glu Glu Lys Trp Ser His Met Gln Thr Phe465 470 475 480Leu Gln Ser Met Ser Ser Thr Asp Lys Val Ile Val Phe Val Ser Arg 485 490 495Lys Ala Val Ala Asp His Leu Ser Ser Asp Leu Ile Leu Gly Asn Ile 500 505 510Ser Val Glu Ser Leu His Gly Asp Arg Glu Gln Arg Asp Arg Glu Lys 515 520 525Ala Leu Glu Asn Phe Lys Thr Gly Lys Val Arg Ile Leu Ile Ala Thr 530 535 540Asp Leu Ala Ser Arg Gly Leu Asp Val His Asp Val Thr His Val Tyr545 550 555 560Asn Phe Asp Phe Pro Arg Asn Ile Glu Glu Tyr Val His Arg Ile Gly 565 570 575Arg Thr Gly Arg Ala Gly Arg Thr Gly Val Ser Ile Thr Thr Leu Thr 580 585 590Arg Asn Asp Trp Arg Val Ala Ser Glu Leu Ile Asn Ile Leu Glu Arg 595 600 605Ala Asn Gln Ser Ile Pro Glu Glu Leu Val Ser Met Ala Glu Arg Phe 610 615 620Lys Ala His Gln Gln Lys Arg Glu Met Glu Arg Lys Met Glu Arg Pro625 630 635 640Gln Gly Arg Pro Lys Lys Phe His 645301240PRTHuman 30Met Pro Arg Gly Ser Trp Lys Pro Gln Val Cys Thr Gly Thr Asp Met1 5 10 15Lys Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg 20 25 30His Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr 35 40 45Tyr Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu 50 55 60Val Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro65 70 75 80Leu Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn 85 90 95Tyr Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr 100 105 110Pro Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg 115 120 125Ser Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro 130 135 140Gln Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys145 150 155 160Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala 165 170 175Cys His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu 180 185 190Ser Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly 195 200 205Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln 210 215 220Cys Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys225 230 235 240Leu His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu 245 250 255Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly 260 265 270Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr 275 280 285Leu Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn 290 295 300Gln Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser305 310 315 320Lys Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg 325 330 335Glu Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys 340 345 350Lys Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly 355 360 365Asp Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val 370 375 380Phe Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp385 390 395 400Pro Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile 405 410 415Arg Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly 420 425 430Leu Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser 435 440 445Gly Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr 450 455 460Val Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His465 470 475 480Thr Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys 485 490 495His Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln 500 505 510Cys Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu 515 520 525Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His 530 535 540Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr545 550 555 560Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys 565 570 575Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp 580 585 590Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys 595 600 605Gln Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp 610 615 620Lys Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile625 630 635 640Ser Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe 645 650 655Gly Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met 660 665 670Arg Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser 675 680 685Gly Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu 690 695 700Leu Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr705 710 715 720Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala 725 730 735Ile Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile 740 745 750Leu Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser 755 760 765Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln 770 775 780Leu Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly785 790 795 800Arg Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys 805 810 815Gly Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala 820 825 830Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp 835 840 845Phe Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala 850 855 860Asp Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu865 870 875 880Arg Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr 885 890 895Val Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro 900 905 910Ala Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln 915 920 925Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp 930 935 940Met Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu945 950 955 960Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn 965 970 975Glu Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser 980 985 990Leu Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr 995 1000 1005Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly 1010 1015 1020Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg 1025 1030 1035Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu 1040 1045 1050Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser 1055 1060 1065Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu 1070 1075 1080Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser 1085 1090 1095Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val 1100 1105 1110Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro 1115 1120 1125Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro 1130 1135 1140Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu 1145 1150 1155Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly 1160 1165 1170Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala 1175 1180 1185Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp 1190 1195 1200Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro 1205 1210 1215Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr 1220 1225 1230Leu Gly Leu Asp Val Pro Val 1235 12403144PRTHuman 31Met Asn His Pro Gly Leu Phe Leu Phe Leu Gly Leu Thr Phe Ala Val1 5 10 15Gln Leu Leu Leu Leu Val Phe Leu Leu Phe Phe Phe Leu Val Trp Trp 20 25 30Asp Gln Phe Gly Cys Arg Cys Asp Gly Phe Ile Leu 35 4032580PRTHuman 32Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu1 5 10 15Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp 20 25 30Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly 35 40 45Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val 50 55

60Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys65 70 75 80Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala 85 90 95Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln 100 105 110Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala 115 120 125Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe 130 135 140Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp145 150 155 160Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro 165 170 175Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu 180 185 190Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe 195 200 205Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln 210 215 220Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile225 230 235 240Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu 245 250 255Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys 260 265 270Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly 275 280 285Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu 290 295 300Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu305 310 315 320Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys 325 330 335Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser 340 345 350Gln Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp Leu Phe Ile 355 360 365Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu Glu Thr Leu 370 375 380Ser Ser Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser385 390 395 400Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala 405 410 415Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg Tyr 420 425 430Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe Asn Leu His 435 440 445Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser Gln Ile Ile Asp 450 455 460Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro Lys465 470 475 480Gly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly 485 490 495Asp Cys Gly Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly 500 505 510Met Ile Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr 515 520 525Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro 530 535 540Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser545 550 555 560Pro Leu Lys Leu Leu Thr Ser Met Ala Ile Ser Val Val Cys Phe Phe 565 570 575Phe Leu Val His 580331069PRTHuman 33Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser1 5 10 15His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu65 70 75 80Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val145 150 155 160Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg225 230 235 240Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro305 310 315 320Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His385 390 395 400Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser465 470 475 480Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe545 550 555 560Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val625 630 635 640Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile705 710 715 720Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu785 790 795 800Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala865 870 875 880Lys Thr Phe Leu Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr 885 890 895Phe Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe 900 905 910Gly Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val 915 920 925Asn Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu 930 935 940Gln Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln945 950 955 960Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr 965 970 975Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser 980 985 990Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln 995 1000 1005Trp Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg 1010 1015 1020Val Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr 1025 1030 1035Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu 1040 1045 1050Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu 1055 1060 1065Asp34404PRTHuman 34Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile Arg Ser1 5 10 15Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Gln Thr 20 25 30Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val Lys Arg Glu Asn Ala 35 40 45Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp Ser Val Ile Pro Lys 50 55 60Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Tyr Ile Cys Glu Glu Asp65 70 75 80Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala Asp Gln Thr Ser Gly 85 90 95Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Val Leu Ser Ser Phe Pro 100 105 110Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly 115 120 125Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile 130 135 140Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser145 150 155 160Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile 165 170 175Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu 180 185 190Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala 195 200 205Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His 210 215 220Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg225 230 235 240Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu 245 250 255Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser 260 265 270Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp 275 280 285Ser Pro Gly Val Val Trp Phe Lys Asp Ser Val Gly Val Ser Gly Asn 290 295 300Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys305 310 315 320Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp 325 330 335Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg 340 345 350Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu 355 360 365Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala 370 375 380Gln Met Pro Thr Thr Glu Arg Val Thr Leu Thr Arg Cys Phe Tyr Leu385 390 395 400Phe Pro Gly His35118PRTHuman 35Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly Tyr Pro Lys Lys Gly1 5 10 15His Gly His Ser Tyr Thr Thr Ala Glu Glu Ala Ala Gly Ile Gly Ile 20 25 30Leu Thr Val Ile Leu Gly Val Leu Leu Leu Ile Gly Cys Trp Tyr Cys 35 40 45Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His Val 50 55 60Gly Thr Gln Cys Ala Leu Thr Arg Arg Cys Pro Gln Glu Gly Phe Asp65 70 75 80His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys Glu Pro Val 85 90 95Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser 100 105 110Pro Pro Pro Tyr Ser Pro 115361255PRTHuman 36Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr1 5 10 15Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly 20 25 30Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser 35 40 45Thr Glu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His 50 55 60Ser Pro Gly Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu65 70 75 80Ala Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gln 85 90 95Asp Val Thr Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr 100 105 110Pro Pro Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro 115 120 125Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 130 135 140Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser145 150 155 160Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 165 170 175Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 180 185 190Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 195 200 205Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 210 215 220Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser225 230 235 240Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 245 250 255Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 260 265 270Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 275 280 285Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 290 295 300Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser305 310 315 320Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 325 330 335Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 340 345 350Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp

Thr Arg Pro Ala Pro 355 360 365Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 370 375 380Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser385 390 395 400Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 405 410 415Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 420 425 430Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 435 440 445Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 450 455 460Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser465 470 475 480Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 485 490 495Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 500 505 510Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 515 520 525Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 530 535 540Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser545 550 555 560Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 565 570 575Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 580 585 590Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 595 600 605Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 610 615 620Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser625 630 635 640Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 645 650 655Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 660 665 670Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 675 680 685Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 690 695 700Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser705 710 715 720Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 725 730 735Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 740 745 750Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 755 760 765Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 770 775 780Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser785 790 795 800Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 805 810 815Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 820 825 830Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 835 840 845Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 850 855 860Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser865 870 875 880Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 885 890 895Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 900 905 910Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 915 920 925Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn 930 935 940Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser945 950 955 960Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly 965 970 975Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe 980 985 990Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His 995 1000 1005Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro 1010 1015 1020Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr 1025 1030 1035Gly Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln 1040 1045 1050Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu 1055 1060 1065Leu Gln Arg Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln 1070 1075 1080Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser 1085 1090 1095Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn 1100 1105 1110Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys Thr Glu Ala 1115 1120 1125Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val Ser Asp 1130 1135 1140Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro Gly 1145 1150 1155Trp Gly Ile Ala Leu Leu Val Leu Val Cys Val Leu Val Ala Leu 1160 1165 1170Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys Gln Cys Arg Arg 1175 1180 1185Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg Asp Thr Tyr 1190 1195 1200His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly Arg Tyr 1205 1210 1215Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val Ser 1220 1225 1230Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val 1235 1240 1245Ala Ala Thr Ser Ala Asn Leu 1250 125537317PRTHuman 37Met Pro Ala Leu Gly Ser Pro Arg Arg Leu Leu Gly Ser Leu Asn Cys1 5 10 15Thr Pro Pro Ala Thr Leu Pro Leu Thr Leu Ala Pro Asn Arg Thr Gly 20 25 30Pro Gln Cys Leu Glu Val Ser Ile Pro Asp Gly Leu Phe Leu Ser Leu 35 40 45Gly Leu Val Ser Leu Val Glu Asn Val Leu Val Val Ala Ala Ile Ala 50 55 60Lys Asn Arg Asn Leu His Ser Pro Met Tyr Tyr Phe Ile Cys Cys Leu65 70 75 80Ala Met Ser Asp Leu Leu Val Ser Val Ser Asn Val Leu Glu Thr Ala 85 90 95Val Met Leu Leu Leu Glu Ala Gly Val Leu Ala Thr Arg Ala Ala Val 100 105 110Val Gln Gln Leu Asp Asn Val Ile Asp Val Leu Ile Cys Ser Ser Met 115 120 125Val Ser Ser Leu Cys Phe Leu Gly Ala Ile Ala Val Asp Arg Tyr Ile 130 135 140Ser Ile Phe Tyr Ala Leu Arg Tyr His Ser Val Val Thr Leu Pro Arg145 150 155 160Ala Trp Arg Ile Ile Ala Ala Ile Trp Val Ala Ser Ile Leu Thr Ser 165 170 175Val Leu Ser Ile Thr Tyr Tyr Asn His Thr Val Val Leu Leu Cys Leu 180 185 190Val Gly Phe Phe Ile Ala Met Leu Ala Leu Met Ala Val Leu Tyr Val 195 200 205His Met Leu Ala Arg Ala Cys Gln His Ala Arg Gly Ile Ala Arg Leu 210 215 220Gln Lys Arg Gln Arg Pro Ile His Gln Gly Phe Gly Leu Lys Gly Ala225 230 235 240Ala Thr Leu Thr Ile Leu Leu Gly Val Phe Phe Leu Cys Trp Gly Pro 245 250 255Phe Phe Leu His Leu Ser Leu Ile Val Leu Cys Pro Gln His Pro Thr 260 265 270Cys Gly Cys Ile Phe Lys Asn Phe Asn Leu Phe Leu Ala Leu Ile Ile 275 280 285Cys Asn Ala Ile Val Asp Pro Leu Ile Tyr Ala Phe Arg Ser Gln Glu 290 295 300Leu Arg Lys Thr Leu Gln Glu Val Leu Gln Cys Ser Trp305 310 31538683PRTHuman 38Met Ser Ser Gly Thr Met Lys Phe Asn Gly Tyr Leu Arg Val Arg Ile1 5 10 15Gly Glu Ala Val Gly Leu Gln Pro Thr Arg Trp Ser Leu Arg His Ser 20 25 30Leu Phe Lys Lys Gly His Gln Leu Leu Asp Pro Tyr Leu Thr Val Ser 35 40 45Val Asp Gln Val Arg Val Gly Gln Thr Ser Thr Lys Gln Lys Thr Asn 50 55 60Lys Pro Thr Tyr Asn Glu Glu Phe Cys Ala Asn Val Thr Asp Gly Gly65 70 75 80His Leu Glu Leu Ala Val Phe His Glu Thr Pro Leu Gly Tyr Asp His 85 90 95Phe Val Ala Asn Cys Thr Leu Gln Phe Gln Glu Leu Leu Arg Thr Thr 100 105 110Gly Ala Ser Asp Thr Phe Glu Gly Trp Val Asp Leu Glu Pro Glu Gly 115 120 125Lys Val Phe Val Val Ile Thr Leu Thr Gly Ser Phe Thr Glu Ala Thr 130 135 140Leu Gln Arg Asp Arg Ile Phe Lys His Phe Thr Arg Lys Arg Gln Arg145 150 155 160Ala Met Arg Arg Arg Val His Gln Ile Asn Gly His Lys Phe Met Ala 165 170 175Thr Tyr Leu Arg Gln Pro Thr Tyr Cys Ser His Cys Arg Glu Phe Ile 180 185 190Trp Gly Val Phe Gly Lys Gln Gly Tyr Gln Cys Gln Val Cys Thr Cys 195 200 205Val Val His Lys Arg Cys His His Leu Ile Val Thr Ala Cys Thr Cys 210 215 220Gln Asn Asn Ile Asn Lys Val Asp Ser Lys Ile Ala Glu Gln Arg Phe225 230 235 240Gly Ile Asn Ile Pro His Lys Phe Ser Ile His Asn Tyr Lys Val Pro 245 250 255Thr Phe Cys Asp His Cys Gly Ser Leu Leu Trp Gly Ile Met Arg Gln 260 265 270Gly Leu Gln Cys Lys Ile Cys Lys Met Asn Val His Ile Arg Cys Gln 275 280 285Ala Asn Val Ala Pro Asn Cys Gly Val Asn Ala Val Glu Leu Ala Lys 290 295 300Thr Leu Ala Gly Met Gly Leu Gln Pro Gly Asn Ile Ser Pro Thr Ser305 310 315 320Lys Leu Val Ser Arg Ser Thr Leu Arg Arg Gln Gly Lys Glu Ser Ser 325 330 335Lys Glu Gly Asn Gly Ile Gly Val Asn Ser Ser Asn Arg Leu Gly Ile 340 345 350Asp Asn Phe Glu Phe Ile Arg Val Leu Gly Lys Gly Ser Phe Gly Lys 355 360 365Val Met Leu Ala Arg Val Lys Glu Thr Gly Asp Leu Tyr Ala Val Lys 370 375 380Val Leu Lys Lys Asp Val Ile Leu Gln Asp Asp Asp Val Glu Cys Thr385 390 395 400Met Thr Glu Lys Arg Ile Leu Ser Leu Ala Arg Asn His Pro Phe Leu 405 410 415Thr Gln Leu Phe Cys Cys Phe Gln Thr Pro Asp Arg Leu Phe Phe Val 420 425 430Met Glu Phe Val Asn Gly Gly Asp Leu Met Phe His Ile Gln Lys Ser 435 440 445Arg Arg Phe Asp Glu Ala Arg Ala Arg Phe Tyr Ala Ala Glu Ile Ile 450 455 460Ser Ala Leu Met Phe Leu His Asp Lys Gly Ile Ile Tyr Arg Asp Leu465 470 475 480Lys Leu Asp Asn Val Leu Leu Asp His Glu Gly His Cys Lys Leu Ala 485 490 495Asp Phe Gly Met Cys Lys Glu Gly Ile Cys Asn Gly Val Thr Thr Ala 500 505 510Thr Phe Cys Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile Leu Gln Glu 515 520 525Met Leu Tyr Gly Pro Ala Val Asp Trp Trp Ala Met Gly Val Leu Leu 530 535 540Tyr Glu Met Leu Cys Gly His Ala Pro Phe Glu Ala Glu Asn Glu Asp545 550 555 560Asp Leu Phe Glu Ala Ile Leu Asn Asp Glu Val Val Tyr Pro Thr Trp 565 570 575Leu His Glu Asp Ala Thr Gly Ile Leu Lys Ser Phe Met Thr Lys Asn 580 585 590Pro Thr Met Arg Leu Gly Ser Leu Thr Gln Gly Gly Glu His Ala Ile 595 600 605Leu Arg His Pro Phe Phe Lys Glu Ile Asp Trp Ala Gln Leu Asn His 610 615 620Arg Gln Ile Glu Pro Pro Phe Arg Pro Arg Ile Lys Ser Arg Glu Asp625 630 635 640Val Ser Asn Phe Asp Pro Asp Phe Ile Lys Glu Glu Pro Val Leu Thr 645 650 655Pro Ile Asp Glu Gly His Leu Pro Met Ile Asn Gln Asp Glu Phe Arg 660 665 670Asn Phe Ser Tyr Val Ser Pro Glu Leu Gln Pro 675 68039376PRTHuman 39Met Met Ala Ser Tyr Pro Glu Pro Glu Asp Ala Ala Gly Ala Leu Leu1 5 10 15Ala Pro Glu Thr Gly Arg Thr Val Lys Glu Pro Glu Gly Pro Pro Pro 20 25 30Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Thr Ala Pro Glu Lys 35 40 45Pro Asp Pro Ala Gln Lys Pro Pro Tyr Ser Tyr Val Ala Leu Ile Ala 50 55 60Met Ala Ile Arg Glu Ser Ala Glu Lys Arg Leu Thr Leu Ser Gly Ile65 70 75 80Tyr Gln Tyr Ile Ile Ala Lys Phe Pro Phe Tyr Glu Lys Asn Lys Lys 85 90 95Gly Trp Gln Asn Ser Ile Arg His Asn Leu Ser Leu Asn Glu Cys Phe 100 105 110Ile Lys Val Pro Arg Glu Gly Gly Gly Glu Arg Lys Gly Asn Tyr Trp 115 120 125Thr Leu Asp Pro Ala Cys Glu Asp Met Phe Glu Lys Gly Asn Tyr Arg 130 135 140Arg Arg Arg Arg Met Lys Arg Pro Phe Arg Pro Pro Pro Ala His Phe145 150 155 160Gln Pro Gly Lys Gly Leu Phe Gly Ala Gly Gly Ala Ala Gly Gly Cys 165 170 175Gly Val Ala Gly Ala Gly Ala Asp Gly Tyr Gly Tyr Leu Ala Pro Pro 180 185 190Lys Tyr Leu Gln Ser Gly Phe Leu Asn Asn Ser Trp Pro Leu Pro Gln 195 200 205Pro Pro Ser Pro Met Pro Tyr Ala Ser Cys Gln Met Ala Ala Ala Ala 210 215 220Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Pro Gly Ser Pro Gly225 230 235 240Ala Ala Ala Val Val Lys Gly Leu Ala Gly Pro Ala Ala Ser Tyr Gly 245 250 255Pro Tyr Thr Arg Val Gln Ser Met Ala Leu Pro Pro Gly Val Val Asn 260 265 270Ser Tyr Asn Gly Leu Gly Gly Pro Pro Ala Ala Pro Pro Pro Pro Pro 275 280 285His Pro His Pro His Pro His Ala His His Leu His Ala Ala Ala Ala 290 295 300Pro Pro Pro Ala Pro Pro His His Gly Ala Ala Ala Pro Pro Pro Gly305 310 315 320Gln Leu Ser Pro Ala Ser Pro Ala Thr Ala Ala Pro Pro Ala Pro Ala 325 330 335Pro Thr Ser Ala Pro Gly Leu Gln Phe Ala Cys Ala Arg Gln Pro Glu 340 345 350Leu Ala Met Met His Cys Ser Tyr Trp Asp His Asp Ser Lys Thr Gly 355 360 365Ala Leu His Ser Arg Leu Asp Leu 370 375

Claims

1.-20. (canceled)

21. A pharmaceutical composition comprising: (a) a population of modified human monocytes isolated from a blood sample from a human cancer patient by isolating cells expressing CD14, wherein the modified human monocytes comprise a chimeric antigen receptor (CAR), and (b) a pharmaceutically acceptable excipient.

22. The pharmaceutical composition of claim 21, wherein the CAR comprises an antigen binding domain, a transmembrane domain and an intracellular domain of a stimulatory and/or co-stimulatory molecule.

23. The pharmaceutical composition of claim 21, wherein the antigen binding domain comprises an anti-HER2 antigen binding domain.

24. The pharmaceutical composition of claim 21, wherein the antigen binding domain of the CAR comprises an antibody selected from the group consisting of a chimeric antibody, synthetic antibody, humanized antibody, single heavy chain antibody, single light chain antibody, scFv, and antigen-binding fragments thereof.

25. The pharmaceutical composition of claim 21, wherein the transmembrane domain of the CAR comprises a CD8 transmembrane domain.

26. The pharmaceutical composition of claim 21, wherein the intracellular domain of the CAR comprises CD80 and/or CD86 and/or CD40L and/or OX40L signaling domains.

27. The pharmaceutical composition of claim 23, wherein the intracellular domain of the CAR comprises a CD3 zeta intracellular domain.

28. The pharmaceutical composition of claim 21, wherein the population of modified human monocytes are isolated from a blood sample from a human cancer patient by isolating cells expressing CD14 by magnetic activated cell sorting (MACS) or fluorescent activated cell sorting (FACS).

29. The pharmaceutical composition of claim 21, wherein the modified human monocytes exhibit targeted effector activity.

30. The pharmaceutical composition of claim 29, wherein the targeted effector activity is directed against a target cell comprising an antigen that specifically binds the antigen binding domain of the CAR.

31. The pharmaceutical composition of claim 29, wherein the targeted effector activity is selected from the group consisting of phagocytosis, cytotoxicity, and antigen presentation.

32. The pharmaceutical composition of claim 29, wherein the targeted effector activity is enhanced by inhibition of CD47 or signal-regulatory protein a activity.

33. The pharmaceutical composition of claim 21, further comprising an agent, wherein the agent is selected from the group consisting of a nucleic acid, a peptide and any combination thereof.

34. The pharmaceutical composition of claim 21, wherein >75% of the modified cells in the pharmaceutical composition express the CAR or wherein the modified human monocytes in the population of cells are genetically modified to stably express the CAR.

35. A method for stimulating an immune response to a target tumor cell or tumor tissue in a subject comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of claim 21.

36. A method of modifying a population of human cells, comprising: isolating monocytes from a blood sample from a human cancer patient and introducing a chimeric antigen receptor (CAR) into the monocytes isolated from the blood sample, wherein isolating monocytes comprises isolating cells expressing CD14.

37. The method of claim 36, wherein introducing the CAR into the monocytes isolated from the blood sample comprises introducing a nucleic acid sequence encoding the CAR.

38. The method of claim 37, wherein introducing the nucleic acid sequence comprises introducing an mRNA encoding the CAR into the monocytes isolated from the blood sample.

39. The method of claim 37, wherein introducing the nucleic acid sequence comprises transducing the monocytes isolated from the blood sample with a viral vector comprising a nucleic acid sequence encoding the CAR.

40. The method of claim 36, wherein the CAR comprises an antigen binding domain, a transmembrane domain and an intracellular domain of a stimulatory and/or co-stimulatory molecule.

41. The method of claim 40, wherein the antigen binding domain comprises an anti-HER2 antigen binding domain.

42. The method of claim 40, wherein the transmembrane domain of the CAR comprises a CD8 transmembrane domain.

43. The method of claim 40, wherein the intracellular domain of the CAR comprises CD80 and/or CD86 and/or CD40L and/or OX40L signaling domains.

44. The method of claim 40, wherein the intracellular domain of the CAR comprises a CD3 zeta intracellular domain.

45. The method of claim 36, wherein isolating monocytes comprises isolating cells expressing CD14 by magnetic activated cell sorting (MACS) or fluorescent activated cell sorting (FACS).

46. The method of claim 36, wherein the monocytes isolated from the blood sample into which the CAR was introduced exhibit targeted effector activity.

47. The method of claim 46, wherein the targeted effector activity is directed against a target cell comprising an antigen that specifically binds the antigen binding domain of the CAR.

48. The method of claim 46, wherein the targeted effector activity is selected from the group consisting of phagocytosis, cytotoxicity, and antigen presentation.

49. A modified M1 macrophage or monocyte comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain and an intracellular domain of a stimulatory and/or co-stimulatory molecule, and wherein the modified M1 macrophage or monocyte comprises an upregulated M1 marker.

50. The modified M1 macrophage or monocyte of claim 49, wherein the upregulated M1 marker is HLA DR.

51. The modified M1 macrophage or monocyte of claim 49, wherein the M1 macrophage or monocyte is not an M2 macrophage or monocyte.

52. The modified M1 macrophage or monocyte of claim 49, wherein the antigen binding domain of the CAR comprises an antibody selected from the group consisting of a chimeric antibody, synthetic antibody, humanized antibody, single heavy chain antibody, single light chain antibody, scFv, and antigen-binding fragments thereof.

53. The modified M1 macrophage or monocyte of claim 49, wherein the transmembrane domain of the CAR comprises a CD8 transmembrane domain.

54. The modified M1 macrophage or monocyte of claim 49, wherein the intracellular domain of the CAR comprises CD80 and/or CD86 and/or CD40L and/or OX40L signaling domains.

55. The modified M1 macrophage or monocyte of claim 49, wherein the intracellular domain of the CAR comprises a CD3 zeta intracellular domain.

56. The modified M1 macrophage or monocyte of claim 49, wherein the modified cell exhibits targeted effector activity.

57. The modified M1 macrophage or monocyte of claim 56, wherein the targeted effector activity is directed against a target cell comprising an antigen that specifically binds the antigen binding domain of the CAR.

58. The modified M1 macrophage or monocyte of claim 56, wherein the targeted effector activity is selected from the group consisting of phagocytosis, cytotoxicity, and antigen presentation.

59. The modified M1 macrophage or monocyte of claim 49, wherein the targeted effector activity is enhanced by inhibition of CD47 or signal-regulatory protein a activity.

60. The modified M1 macrophage or monocyte of claim 49, further comprising an agent, wherein the agent is selected from the group consisting of a nucleic acid, a peptide and any combination thereof.

61. The modified M1 macrophage or monocyte of claim 49, wherein the modified cell is genetically modified to express the CAR.

62. A pharmaceutical composition comprising the modified M1 macrophage or monocyte of claim 49 and a pharmaceutically acceptable carrier.

63. The pharmaceutical composition of claim 62, wherein >75% of the modified cells in the pharmaceutical composition express the CAR or wherein the modified human monocytes in the population of cells are genetically modified to stably express the CAR.

64. A method for stimulating an immune response to a target tumor cell or tumor tissue in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the modified M1 macrophage or monocyte of claim 49.