VCX / Y peptides and uses thereof

Abstract

Methods and compositions related to peptides from the Variable Charge X-Linked / Y-linked (VCX / Y) family (e.g., VCX1, VCX2, VCX3A, VCX3B, VCY) are provided. [Solution] Provided herein are tumor antigen VCX / Y-specific peptides and engineered VCX / Y-specific T cell receptors. Also provided herein are methods for generating VCX / Y-specific immune cells and their use for the treatment of cancer. Furthermore, VCX / Y-specific peptides can be used as vaccines.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 62 / 910,128, filed October 3, 2019, which is incorporated by reference in its entirety. The present disclosure relates generally to the fields of immunology and medicine. More specifically, the present disclosure relates to tumor antigen peptides and their use for treating cancer. [Background technology]

[0002] Various T cell-based therapies have shown remarkable promise as a method for treating many cancers. Unfortunately, however, this approach is also hampered by the paucity of immunogenic antigen targets for common cancers and potential toxicity to non-cancerous tissues. These T cell-based therapies include ACT (adoptive cell transfer) and vaccination approaches. ACT generally involves injecting a large number of autologous activated tumor-specific T cells into patients to treat, for example, cancer. ACT has produced therapeutic clinical responses in melanoma patients (Non-Patent Document 1: Yee, 2002; Dudley, 2002; Yee, 2014). In general, three steps are usually required to develop an effective anti-tumor T cell response: antigen-priming and activation of antigen-specific T cells, migration of activated T cells to the tumor site, and tumor recognition and killing by antigen-specific T cells. Selection of target antigen is important to induce effective antigen-specific T cells.

[0003] Although several tumor-associated antigens have been identified for melanoma and a few other solid tumor malignancies, few immunogenic targets are known for pancreatic, ovarian, gastric, lung, cervical, breast, and head and neck cancers. There is a strong rationale for identifying and validating novel epitopes and target antigens for these common and difficult-to-treat malignancies. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yee, 2002; Dudley, 2002; Yee, 2014 Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides, at least in some embodiments, methods and compositions related to peptides from the Variable Charge X-Linked / Y-linked (VCX / Y) family (e.g., VCX1, VCX2, VCX3A, VCX3B, VCY), including peptides that can be used in adoptive T cell therapy. In some embodiments, the peptides of the present invention may be used to expand VCX / Y-specific T cells in vitro, which are administered to a mammalian subject, such as a human patient, to treat a disease (e.g., cancer). In further embodiments, T cells are engineered to express a T cell receptor (TCR) with antigen specificity for VCX / Y. In other embodiments, the peptides of the present invention may be administered to a mammalian subject to induce an immune response or to vaccinate the mammalian subject against the peptide, which may be useful for treating a disease (e.g., cancer, etc.) or reducing the risk of contracting or relapsing from a disease (e.g., cancer, etc.).

[0006] In one embodiment, the disclosure provides an isolated VCX / Y (e.g., VCX3A) peptide of 35 amino acids or less in length that comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 (SEVEEPLSQ). In some aspects, the peptide comprises an amino acid sequence having at least 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or 100 percent sequence identity to SEQ ID NO: 1. In certain embodiments, the peptide is capable of inducing cytotoxic T lymphocytes (CTLs).

[0007] In certain aspects, the peptide is 30 amino acids or less in length, such as 29 amino acids, 28 amino acids, 27 amino acids, 26 amino acids, 25 amino acids, 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids, 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, or 10 amino acids in length.

[0008] In another embodiment, a pharmaceutical composition is provided that comprises the isolated VCX / Y peptide of the above embodiment and a pharmaceutical carrier.In some aspects, the pharmaceutical composition is formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation or subcutaneous injection, by way of example only.In certain aspects, the peptide is included in a liposome, nanoparticle (e.g., lipid-containing nanoparticle), or lipid-based carrier.In some aspects, the pharmaceutical preparation is formulated for injection or inhalation as a nasal spray.

[0009] Further embodiments provide isolated nucleic acids encoding the VCX / Y peptides of the above embodiments. Also provided herein are vectors comprising a contiguous sequence of nucleic acid encoding a VCX / Y peptide or a contiguous sequence comprising nucleic acid encoding a VCX / Y peptide.

[0010] In yet another embodiment, a method for promoting immune response in a subject is provided, comprising administering an effective amount of the VCX / Y peptide of the above embodiment to the subject, wherein the peptide induces antigen-specific T cells in the subject.In some aspects, the subject is, for example, diagnosed with cancer or at risk for cancer, including at higher risk than the general population.In certain aspects, the cancer is pancreatic cancer, ovarian cancer, gastric cancer or breast cancer.In certain aspects, the subject is a human.

[0011] In further aspects, the method further comprises administering at least a second anticancer therapy.In some aspects, the second anticancer therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy or surgery.In certain aspects, the immunotherapy is an immune checkpoint inhibitor.In one specific aspect, the immune checkpoint inhibitor is an anti-PD1 monoclonal antibody.

[0012] Further embodiments provide a method of producing VCX / Y-specific T cells, comprising obtaining a starting population of T cells and contacting the starting population of T cells with a VCX / Y peptide of the above embodiment, thereby generating VCX / Y-specific T cells. In some aspects, the contacting is further defined as co-culturing the starting population of T cells with an antigen-presenting cell (APC) that presents the VCX / Y peptide of the above embodiment on its surface. In certain aspects, the APC is a dendritic cell. In some aspects, the starting population of T cells is a CD8 + The T cell is a T cell. In certain aspects, the T cell is a cytotoxic T lymphocyte (CTL). In some aspects, the obtaining comprises isolating a starting population of T cells from peripheral blood mononuclear cells (PBMCs). Also provided herein is a pharmaceutical composition comprising the VCX / Y-specific T cell produced by the method herein.

[0013] Yet further embodiments provide antigen receptors having antigen specificity for VCX / Y, such as T cell receptors (TCRs) or chimeric antigen receptors (CARs), etc. Another embodiment provides T cells engineered to express a VCX / Y-specific TCR and / or a VCX / Y-specific CAR.

[0014] Another embodiment provides a method of treating cancer in a subject, comprising administering an effective amount of the VCX / Y-specific T cells of the above embodiment to the subject. In some aspects, the cancer is thymoma, bladder cancer, uterine cancer, melanoma, sarcoma, cervical cancer or head and neck cancer. In certain aspects, the subject is a human. In some aspects, the cells are autologous or allogeneic with respect to the recipient individual. In some aspects, the subject is determined to have cancer cells that express VCX / Y, but in other cases, it is unknown whether the subject has cancer cells that express VCX / Y.

[0015] In some aspects, the host cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), induced pluripotent stem (iPS) cells, or mixtures thereof. In certain aspects, the host cells are isolated from the umbilical cord. In some aspects, the host cells are autologous or allogeneic with respect to the recipient individual. In certain aspects, the T cells are CD8 + T cells, CD4 + T cells, γδ T cells, or a mixture thereof.

[0016] In certain aspects, the method further comprises lymphodepletion of the subject prior to administration of the antigen-specific T cells. In some aspects, lymphodepletion comprises administration of cyclophosphamide and / or fludarabine.

[0017] In some aspects, the method further comprises administering at least a second therapeutic agent. In certain aspects, the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy and / or biological therapy. In certain aspects, the immunotherapy is one or more immune checkpoint inhibitors. In some specific aspects, the immune checkpoint inhibitor is an anti-PD1 monoclonal antibody.

[0018] In certain aspects, the VCX / Y-specific T cells and / or at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, topically, or by direct injection or perfusion.

[0019] In some aspects, the subject is determined to have cancer cells that express a protein of the VCX / Y family. In certain aspects, the protein is VCX3A.

[0020] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The subject matter of the present disclosure may be better understood by reference to one or more of these drawings in combination with the description of exemplary embodiments presented herein. [Brief description of the drawings]

[0022] [Figure 1]As an example, screening of an overlapping peptide library for a target peptide recognized by VCX54 TCR-T, the sequence of which in its entirety is SEQ ID NO:7.

[0023] [Diagram 2] VCX118 peptide cross-reactivity detection.

[0024] [Diagram 3] Peptide titration killing assay.

[0025] [Figure 4] VCX118 peptide HLA-A2 binding assay. In three separate column configurations, from left to right, the columns represent 100ug, 30ug and 10ug.

[0026] [Diagram 5] VCX54 TCR-T recognition against longer peptides of different lengths, including VCX118. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] For individuals with many different cancer types, various T cell-based immunotherapies have become promising approaches with proven efficacy. However, antigen-specific T cell therapy for most cancer types is not feasible due to the lack of currently known tumor-associated antigens, which has led to their stalled clinical development. Research in this disclosure has identified novel VCX / Y family-derived peptide epitopes found in all VCX / Y family members, including VCX1, VCX2, VCX3A, VCX3B and VCY. These peptide epitopes have been used to generate antigen-specific cytotoxic T lymphocytes (CTLs) from patients' peripheral blood mononuclear cells (PBMCs) that recognize endogenously presented antigens on the surface of allogeneic tumor cell lines, thereby causing tumor cell death. Thus, this antigen-specific CTL can be used to target solid cancers (e.g., pancreatic cancer, ovarian cancer, gastric cancer and breast cancer).

[0028] Thus, the present disclosure provides tumor antigen-specific peptides, such as tumor antigen VCX / Y, for use as immunotherapy to treat cancer or associated with therapy to treat cancer. An exemplary VCX / Y peptide, VCX118 (e.g., comprising SEQ ID NO: 1), is disclosed herein, whose sequence is shared with all VCX / Y family members, including VCX1, VCX2, VCX3A, VCX3B and VCY. For example, a tumor antigen-specific peptide may be contacted with or used to stimulate a population of T cells to induce proliferation of T cells that recognize or bind to the tumor antigen-specific peptide. In other embodiments, a VCX / Y-specific peptide of the present disclosure may be administered to a subject, such as a human patient, to enhance the subject's immune response to cancer.

[0029] VCX / Y-specific peptides may be included in active immunotherapy (e.g., cancer vaccines) or passive immunotherapy (e.g., adoptive immunotherapy). Active immunotherapy involves immunizing a subject with one or more purified tumor antigens or one or more immunodominant VCX / Y-specific peptides (native or modified); alternatively, antigen-presenting cells pulsed with VCX / Y-specific peptides (or transfected with genes encoding tumor antigens) may be administered to the subject. VCX / Y-specific peptides may, for example, be modified, i.e., contain one or more mutations, such as substitution mutations, including conservative mutations. Passive immunotherapy includes adoptive immunotherapy. Adoptive immunotherapy generally involves administering cells to a subject, where the cells (e.g., cytotoxic T cells) have been sensitized in vitro to VCX / Y-specific peptides (see, for example, U.S. Pat. No. 7,910,109).

[0030] In particular, the patient's own VCX / Y-specific T cells can be generated ex vivo for effective immune-based therapy within a short period of time, e.g., 6-8 weeks. The T cells can be generated from autologous or allogeneic T cells (e.g., CD4 + T cells, CD8 + The peptides or corresponding encoded polynucleotides may be isolated and expanded, for example, by using tetramer-induced selection and rapid expansion protocol (REP) from T cells. The peptides or corresponding encoded polynucleotides may then be loaded into dendritic cells, LCLs, PBMCs and / or artificial antigen presenting cells (aAPCs) and then co-cultured with T cells by several rounds of stimulation to generate antigen-specific CTL cell lines or clones. Furthermore, the manipulation of immune-modulating parameters may enhance the in vivo effector function and long-term persistence of these expanded antigen-specific T cells. These autologous CTL cells may be used for adoptive immunotherapy for VCX / Y-positive cancer patients. Additionally, other VCX / Y-specific cells that may be generated from the present disclosure include autologous or allogeneic NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs) and / or induced pluripotent stem (iPS) cells. These cells may be isolated from blood, bone marrow, lymph, umbilical cord and / or lymphoid organs. I. Definition

[0031] Consistent with long-standing patent law practice, the words "a" and "an" mean "one or more" when used herein in conjunction with the word comprising, including in the claims. As used in this specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a nucleic acid" includes a plurality of nucleic acids, including mixtures thereof. Some embodiments of the present disclosure may consist of, or consist essentially of, one or more of the elements, method steps and / or methods of the present disclosure. It is contemplated that any method or composition described herein may be practiced with respect to any other method or composition described herein, and that the various embodiments may be combined.

[0032] Throughout this specification, unless the context otherwise requires, the words "comprise", "comprises" and "comprising" shall be understood to mean the inclusion of the specified steps or elements or group of steps or elements but not the exclusion of any other steps or elements or group of steps or elements. By "consisting of", it is meant to include and be limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that other elements may not be present. By "consisting essentially of", it is meant to include whatever elements are recited following this phrase, and to be limited to other elements that neither interfere with nor contribute to the activity or function specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may be present or absent depending on whether they affect the activity or function of the recited elements.

[0033] As used herein, the terms "or" and "and / or" are utilized to describe multiple components in combination or mutually exclusive. For example, "x, y and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically intended that x, y, or z may be specifically excluded from an embodiment.

[0034] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method being employed in each instance to determine the value, or the variation that exists among study subjects.

[0035] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," "a further embodiment," or combinations thereof means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, such particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0036] "Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject, or the administration of a procedure or modality to a subject, for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment can include administering T cell therapy and / or a peptide.

[0037] "Subject" and "patient" and "individual" refer to either humans or non-humans (e.g., primates, mammals, vertebrates, etc.). In certain embodiments, the subject is a human of any sex or age or race.

[0038] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that directly or indirectly promotes or enhances the well-being of a subject with respect to the medical treatment of the condition. This includes, but is not limited to, a reduction in the frequency or severity of one or more signs or symptoms of a disease. For example, the treatment of cancer may involve, for example, a reduction in the size of one or more tumors, a reduction in the invasiveness of one or more tumors, a reduction in the growth rate of the cancer, a reduction in tumor burden, or prevention of metastasis or metastatic spread. The treatment of cancer may also refer to prolonging the survival of a subject with cancer.

[0039] An "anti-cancer" agent can negatively affect cancer cells / tumors in a subject, for example, by promoting the death of cancer cells, inducing apoptosis in cancer cells, slowing the growth rate of cancer cells, reducing the appearance or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the longevity of a subject with cancer.

[0040] The term "antibody" is used herein in the broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

[0041] As used herein, "essentially free" is used herein to mean, with respect to a named component, that the named component is not intentionally incorporated into the composition and / or is present merely as a contaminant or in only trace amounts. Thus, the total amount of the named component resulting from any unintentional incorporation into the composition is well below 0.05%, and preferably less than 0.01%. Most preferred are compositions in which the amount of the named component cannot be detected by standard analytical methods.

[0042] The expression "pharmaceutical or pharmacologically acceptable or pharmacologic acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to an animal (such as, for example, a human) as appropriate. The preparation of pharmaceutical compositions containing an antibody or additional active ingredient will be known to those skilled in the art in light of the present disclosure. Moreover, it will be understood that for administration to an animal (such as a human), the preparation must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0043] As used herein, "pharmaceutical acceptable carriers" include any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate, etc.), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption retardants, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluid and nutritional supplements, such materials, and combinations thereof, as would be known to one skilled in the art. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to widely known parameters.

[0044] The term "unit dose" or "(single) dose" refers to physically separate units suitable for use in a subject, each containing a predetermined amount of a therapeutic composition calculated to produce the desired response in relation to its administration, i.e., in relation to the appropriate route and treatment regimen, discussed above. The amount to be administered is dependent on the desired effect, both according to the number of treatments and the unit dose. The actual (single) dose of the composition of the present embodiment administered to a patient or subject can be determined by various physical and physiological factors, such as the subject's weight, age, health and sex, the type of disease being treated, the degree of disease penetration, previous or concurrent therapeutic interventions, the idiopathic nature of the patient, the route of administration, and the potency, stability and toxicity of the particular therapeutic agent. For example, the dose may also include a range from about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (including such ranges inclusive of doses therebetween) or more, and any range that may be derived therein. In non-limiting examples of ranges that may be derived from the numbers recited herein, ranges of about 5 μg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. may be administered. The person responsible for administration will in any event determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject. In some embodiments, the dose (per dose) of antigen-specific T cell infusion may include about 100 million to about 30 billion cells (e.g., 10 billion, 15 billion, or 20 billion cells, etc.).

[0045] The term "immune checkpoint" refers to a molecule, such as a protein, in the immune system that provides signals to its components to balance immune response. Known immune checkpoint proteins include CTLA-4, PD1 and its ligands PD-L1 and PD-L2, as well as LAG-3, BTLA, B7H3, B7H4, TIM3, KIR. It is recognized in the art that various pathways involving LAG3, BTLA, B7H3, B7H4, TIM3 and KIR constitute immune checkpoint pathways similar to CTLA-4-dependent pathways and PD-1-dependent pathways (see, for example, Pardoll (2012); Mellman et al. (2011)).

[0046] "Immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reducing and completely blocking the function. In particular, the immune checkpoint protein is a human immune checkpoint protein. Thus, the immune checkpoint protein inhibitor is particularly an inhibitor of a human immune checkpoint protein.

[0047] As used herein, a "protective immune response" refers to a response by the immune system of a mammalian host to a cancer. A protective immune response may result in a therapeutic effect for treating the cancer, such as reducing tumor size or increasing survival.

[0048] As used herein, the term "antigen" is a molecule that can be bound by an antibody or a T cell receptor. Antigens may generally be used to induce humoral and / or cellular immune responses that result in the production of B and / or T lymphocytes.

[0049] The terms "tumor-associated antigen," "tumor antigen," and "cancer cell antigen" are used interchangeably herein. In each case, these terms refer to a protein, glycoprotein, or carbohydrate that is specifically or preferentially expressed by cancer cells.

[0050] The term "chimeric antigen receptor (CAR)" as used herein may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, including engineered receptors in which artificial specificity is grafted onto a specific immune effector cell. Various CARs may be used to confer the specificity of a monoclonal antibody to a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In a specific embodiment, the CAR directs the specificity of the cell to, for example, a tumor-associated antigen. In some embodiments, the CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain that includes a tumor-associated antigen binding region. In certain aspects, the CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to CD3-zeta, a transmembrane domain, and one or more endodomains. The specificity of other CAR designs may be derived from the receptor's ligand (e.g., a peptide) or from a pattern recognition receptor (e.g., dectin, etc.). In certain cases, the spacing of antigen recognition domains can be altered to reduce activation-induced cell death.In certain cases, CARs include domains for additional costimulatory signaling, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10 and / or OX40.In some cases, various molecules can be co-expressed with CARs, including costimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that cause conditional elimination of T cells when prodrugs are added, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0051] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" or "homology" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. Alignment and percent homology or percent sequence identity can be determined using a variety of software programs known in the art, for example, using the software programs described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FMA Usubel et al., eds., 1987), Supplement 30, Section 7.7.18, Table 7.7.1. Preferably, default parameters are used for the alignment. A preferred alignment program is BLAST, with default parameters used. In particular, the preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. II. VCX / Y peptide

[0052] Embodiments of the present disclosure relate to tumor antigen-specific peptides, such as peptides derived from the VCX / Y tumor antigen. In certain embodiments, the tumor antigen-specific peptide has the amino acid sequence of the VCX / Y peptide (SEVEEPLSQ; SEQ ID NO:1). The tumor antigen-specific peptide may have an amino acid sequence that has at least 80 percent, 85 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or 100 percent sequence identity with the peptide sequence of SEQ ID NO:1.

[0053] As used herein, the term "peptide" encompasses an amino acid chain comprising 8 to 35 amino acid residues in length, e.g., 8 to 25 amino acids, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids, or any range that may be derivable therein, etc. For example, a VCX / Y peptide of the present disclosure may in some embodiments comprise, consist of, or consist essentially of the VCX118 peptide of SEQ ID NO:1. The peptides of the present disclosure are antigenic peptides in certain embodiments, and as used herein, an "antigenic peptide" is a peptide that, when introduced into a vertebrate, is capable of stimulating the production of antibodies in the vertebrate, i.e., is antigenic, provided that the antibodies can selectively recognize and / or bind to the antigenic peptide. Antigenic peptides may include immunoreactive VCX / Y peptides and may also include additional sequences that may or may not be derived from native antigens and may be heterologous, and such sequences may, but need not, be immunogenic. In certain embodiments, the VCX / Y peptide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length, or any range derivable therein. In specific embodiments, the tumor antigen-specific peptide (e.g., the VCX / Y peptide) is 8 to 35 amino acids in length.In some embodiments, the tumor antigen-specific peptide (e.g., a VCX / Y peptide) is 7-10, 8-10, 9-10, 7-9, 7-8, or 8-9 amino acids in length.

[0054] As will be understood by those skilled in the art, MHC molecules can bind peptides of various sizes, but not typically full-length proteins. Although MHC class I molecules are traditionally described as binding peptides of 8-11 amino acids in length, it has been shown that peptides as long as 15 amino acids can bind to MHC class I molecules by bulging in the center of the binding site or extending outward from the MHC class I binding groove (Guo et al., 1992; Burrows et al., 2006; Samino et al., 2006; Stryhn et al., 2000; Collins et al., 1994; Blanchard and Shastri, 2008). Furthermore, recent studies have also demonstrated that longer peptides can be more efficiently endocytosed, processed, and presented by antigen-presenting cells (Zwaveling et al., 2002; Bijker et al., 2007; Melief and van der Burg, 2008; Quintarelli et al., 2011). As revealed in Zwaveling et al. (2002), peptides up to 35 amino acids in length can be used to selectively bind to class II MHC and are effective. As will be readily understood by those skilled in the art, naturally occurring full-length tumor antigens, such as VCX / Y, will not be useful for selectively binding to class II MHC, as they will be taken up by endocytosis and will cause T cell proliferation. In general, naturally occurring full-length tumor antigen proteins do not exhibit these properties and therefore will not be useful for these immunotherapy purposes.

[0055] In certain embodiments, tumor antigen-specific peptides (e.g., VCX / Y peptides) are immunogenic or antigenic. As shown in the following examples, various tumor antigen-specific peptides (e.g., VCX / Y peptides) of the present disclosure can promote T cell proliferation. It is expected that such peptides can be used to induce some degree of protective immunity.

[0056] The tumor antigen-specific peptide (e.g., VCX / Y peptide) may be a recombinant peptide, a synthetic peptide, a purified peptide, an immobilized peptide, a detectably labeled peptide, an encapsulated peptide, or a vector-expressed peptide (e.g., a peptide encoded by a nucleic acid in a vector comprising a heterologous promoter operably linked to the nucleic acid). In some embodiments, a synthetic tumor antigen-specific peptide (e.g., VCX / Y peptide) may be administered to a subject (e.g., a human patient, etc.) to induce an immune response in the subject. Compared with a recombinantly expressed peptide, a synthetic peptide may exhibit certain advantages, such as a reduced risk of bacterial contamination. A tumor antigen-specific peptide (e.g., VCX / Y peptide) may also be included in a pharmaceutical composition (e.g., a vaccine composition, etc.) formulated for administration to a mammalian subject or a human subject. A. Cell-penetrating peptides

[0057] In some embodiments, immunotherapy may utilize the tumor antigen-specific peptides of the present disclosure (e.g., VCX / Y peptides) associated with cell-penetrating agents such as liposomes or cell-penetrating peptides (CPPs). Antigen-presenting cells (e.g., dendritic cells, etc.) pulsed with peptides may be used to enhance antitumor immunity (Celluzzi et al., 1996; Young et al., 1996). Liposomes and CPPs are described in more detail below. In some embodiments, immunotherapy may utilize nucleic acids encoding the tumor antigen-specific peptides of the present disclosure (e.g., VCX / Y peptides), delivered, for example, in viral or non-viral vectors.

[0058] Tumor antigen-specific peptides (e.g., VCX / Y peptides) may also be associated with or covalently linked to cell-penetrating peptides (CPPs). Cell-penetrating peptides that may be covalently linked to tumor antigen-specific peptides (e.g., VCX / Y peptides) include, for example, HIV Tat, herpes virus VP22, Drosophila Antennapedia homeobox gene product, signal sequence, fusion sequence, or protegrin I. Covalently linking a peptide to a CPP can prolong the presentation of the peptide by dendritic cells, thus enhancing anti-tumor immunity (Wang and Wang, 2002). In some embodiments, tumor antigen-specific peptides of the present disclosure (e.g., tumor antigen-specific peptides of the present disclosure that are included within a peptide or polyepitope string) (e.g., VCX / Y peptides) may be covalently linked to a CPP (e.g., via a peptide bond) to generate a fusion protein. In other embodiments, a tumor antigen-specific peptide (e.g., a VCX / Y peptide), or a nucleic acid encoding a tumor antigen-specific peptide, may be encapsulated in or associated with a liposome (e.g., a multilamellar liposome, a vesicular liposome, or a multivesicular liposome, etc.), an exocytosis vesicle, or an exosome.

[0059] As used herein, "association" means a physical association, a chemical association, or both. For example, the association can involve covalent bonding, hydrophobic interactions, inclusion, or surface adsorption, etc.

[0060] As used herein, "cell penetrating agent" refers to a composition or compound that enhances the intracellular delivery of peptide / polyepitope string to antigen-presenting cells. For example, the cell penetrating agent may be a lipid that enhances the ability to cross plasma membrane when associated with peptide. Alternatively, the cell penetrating agent may be a peptide. A variety of cell penetrating peptides (CPPs) are known in the art, including, for example, the Tat protein of HIV (Frankel and Pabo, 1988), the VP22 protein of HSV (Elliott and O'Hare, 1997), and fibroblast growth factor (Lin et al., 1995).

[0061] Cell-penetrating peptides (or "protein transduction domains") have been identified from the Drosophila Antennapedia homeobox gene (Antp), HIV Tat, and the third helix of herpesvirus VP22, all of which contain positively charged domains rich in arginine and lysine residues (Schwarze et al., 2000; Schwarze et al., 1999). Hydrophobic peptides derived from signal sequences have also been identified as cell-penetrating peptides (Rojas et al., 1996; Rojas et al., 1998; Du et al., 1998). Linking these peptides to marker proteins (such as β-galactosidase) has been shown to result in efficient internalization of the marker proteins into cells, and chimeric in-frame fusion proteins containing these peptides have been used to deliver proteins to a wide variety of cell types both in vitro and in vivo (Drin et al., 2002). Fusing these cell-penetrating peptides to a tumor antigen-specific peptide according to the present disclosure (eg, a VCX / Y peptide) may enhance cellular uptake of the polypeptide.

[0062] In some embodiments, cellular uptake is enhanced by linking lipids (such as stearate or myristilate) to the polypeptide. Lipidation has been shown to enhance the passage of peptides into cells. Linking lipid moieties is another way in which the present disclosure increases polypeptide uptake by cells. Cellular uptake is discussed further below.

[0063] The tumor antigen-specific peptide (e.g., VCX / Y peptide) of the present disclosure may be included in a liposomal vaccine composition. For example, the liposomal composition may be or may include a proteoliposome composition. Various methods for producing a proteoliposome composition that may be used with the present disclosure are described, for example, in Neelapu et al. (2007) and Popescu et al. (2007). In some embodiments, the proteoliposome composition may be used to treat melanoma.

[0064] By enhancing the uptake of tumor antigen-specific polypeptides, it may be possible to reduce the amount of protein or peptide required for treatment. This can result in a significant reduction in treatment costs and increased supply of therapeutic agents. A lower (per dose) dosage can also minimize potential immunogenicity of peptides and limit toxic side effects.

[0065] In some embodiments, the tumor antigen-specific peptide (e.g., VCX / Y peptide) may be associated with the nanoparticle to form a nanoparticle-polypeptide complex. In some embodiments, the nanoparticle is a liposome or other lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP: cholesterol vesicle). In other embodiments, the nanoparticle is an iron oxide-based superparamagnetic nanoparticle. Superparamagnetic nanoparticles, with diameters in the range of about 10-100 nm, are small enough to avoid capture by the spleen, but large enough to avoid clearance by the liver. Particles of this size can enter very small capillaries and be effectively distributed in body tissues. The superparamagnetic nanoparticle-polypeptide complex can be used as an MRI contrast agent to identify and track those cells that take up the tumor antigen-specific peptide (e.g., VCX / Y peptide). In some embodiments, the nanoparticle is a semiconductor nanocrystal or a semiconductor quantum dot, both of which can be used in optical imaging. In further embodiments, the nanoparticle can be a nanoshell that includes a gold layer over a silica core. One advantage of nanoshells is that polypeptides can be conjugated to the gold layer using standard chemistry. In other embodiments, the nanoparticles can be fullerenes or nanotubes (Gupta et al., 2005).

[0066] Peptides are quickly removed from circulation by the kidney and are also susceptible to degradation by proteases in serum. By associating tumor antigen-specific peptides (e.g., VCX / Y peptides) with nanoparticles, the nanoparticle-polypeptide complexes of the present disclosure may provide protection from degradation and / or reduced clearance by the kidney. This may increase the serum half-life of the polypeptide, thereby reducing the polypeptide dose required for effective treatment. Furthermore, this may reduce the cost of treatment and minimize immunological problems and toxic reactions of the treatment. B. Polyepitope string

[0067] In some embodiments, the tumor antigen-specific peptide (e.g., VCX / Y peptide) is included or is included in a polyepitope string. A polyepitope string is a peptide or polypeptide that contains multiple antigenic epitopes from one or more antigens linked together. A polyepitope string may be used to induce an immune response in a subject, such as in a human subject. Various polyepitope strings have been used previously to target malaria and other pathogens (Baraldo et al., 2005; Moorthy et al., 2004; Baird et al., 2004). A polyepitope string may represent a nucleic acid (e.g., a nucleic acid that encodes multiple antigens, including VCX / Y peptides), or a peptide or polypeptide (e.g., a peptide or polypeptide that contains multiple antigens, including VCX / Y peptides). A polyepitope string may be included in a cancer vaccine composition. C. Biologically functional equivalents

[0068] The tumor antigen-specific peptides (e.g., VCX / Y peptides) of the present disclosure may be modified to contain various amino acid substitutions, insertions, and / or deletions that do not alter their respective interactions. Such biologically functional equivalents of tumor antigen-specific peptides (e.g., VCX / Y peptides) may be molecules with similar or otherwise desirable characteristics. As a non-limiting example, certain amino acids may be substituted for other amino acids in the tumor antigen-specific peptides (e.g., VCX / Y peptides) disclosed herein without appreciable loss of interaction capacity. In some embodiments, the tumor antigen-specific peptides have substitution mutations at anchor residues, such as substitution mutations at one, two, or all of positions 1 (P1), 2 (P2), and / or 9 (P9). Thus, tumor antigen-specific peptides (e.g., VCX / Y peptides) disclosed herein (or nucleic acids encoding such peptides) that are modified in sequence and / or structure but not altered in biological utility or activity are still intended to be within the scope of the compositions and methods disclosed herein.

[0069] It is also well understood by those skilled in the art that the definition of a biologically functional equivalent peptide entails the notion that there is a limit to the number of changes that can be made within a defined portion of a molecule while still maintaining an acceptable level of equivalent biological activity. Thus, a biologically functional equivalent peptide is defined herein as such a peptide in which some, but not most or all, of the amino acids can be substituted. Of course, multiple different peptides with various substitutions can be easily made and used according to the present disclosure.

[0070] Those skilled in the art also recognize that if a particular residue is shown to be particularly important for the biological or structural properties of a peptide (e.g., a residue in a particular epitope), such residue generally cannot be replaced. This may be the case in the present disclosure, since mutations in the tumor antigen-specific peptides disclosed herein (e.g., VCX / Y peptides) may result in loss of species specificity, thereby reducing the usefulness of the resulting peptide for use in the methods of the present disclosure. Thus, it is understood that peptides that are antigenic and contain conservative amino acid substitutions are included in the present disclosure. Conservative substitutions are least likely to dramatically change the activity of a protein. "Conservative amino acid substitution" refers to the replacement of an amino acid with a chemically similar amino acid, i.e., the replacement of a non-polar amino acid with another non-polar amino acid, for example, the replacement of a polar amino acid with another polar amino acid, the replacement of an acidic residue with another acidic amino acid, etc.

[0071] Amino acid substitutions, such as those that may be used in modifying the tumor antigen-specific peptides (e.g., VCX / Y peptides) disclosed herein, are generally based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge and size. Analysis of the size, shape and type of amino acid side chain substituents reveals that arginine, lysine and histidine are all positively charged residues, alanine, glycine and serine are all similar in size, and phenylalanine, tryptophan and tyrosine all have generally similar shapes. Thus, based on these considerations, arginine, lysine and histidine, as well as alanine, glycine and serine, as well as phenylalanine, tryptophan and tyrosine, are defined herein as biologically functional equivalents. In some embodiments, the mutations may enhance TCR-pMHC interaction and / or peptide-MHC binding.

[0072] The present disclosure also contemplates isoforms of the tumor antigen-specific peptides (e.g., VCX / Y peptides) disclosed herein. Isoforms contain the same number and types of amino acids as the peptides of the present disclosure, but have different molecular structures. The isoforms contemplated by the present disclosure are isoforms that have the same properties as the peptides of the present disclosure as described herein.

[0073] Non-standard amino acids may be incorporated into proteins by chemical modification of existing amino acids or by de novo synthesis of the peptides disclosed herein. A non-standard amino acid refers to an amino acid whose chemical structure differs from the 20 standard amino acids encoded by the genetic code.

[0074] In selected embodiments, the present disclosure contemplates chemical derivatives of the tumor antigen-specific peptides disclosed herein (e.g., VCX / Y peptides). "Chemical derivative" refers to peptides that have one or more residues that are chemically derivatized by reaction of a functional side group and that retain biological activity and utility. Such derivatized peptides include, for example, those in which free amino groups are derivatized to form specific salts, or those that are derivatized by alkylation and / or acylation, among others, with p-toluenesulfonyl, carbobenzoxy, t-butylocycarbonyl, chloroacetyl, formyl, or acetyl groups. Free carboxyl groups may be derivatized to form organic or inorganic salts, methyl and ethyl esters or other types of esters or hydrazides, and preferably amides (primary or secondary). Chemical derivatives may include those peptides that contain one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline may be used in place of serine, and ornithine may be used in place of lysine.

[0075] It should be noted that all amino acid residue sequences are represented herein by formulas whose left-to-right orientation is the conventional direction from amino terminus to carboxy terminus. It should further be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues. The amino acids described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be used in place of any L-amino acid residue, so long as the desired functional properties as set forth herein are retained by the protein. D. Nucleic Acids Encoding Tumor Antigen-Specific Peptides

[0076] In one aspect, the present disclosure provides a nucleic acid encoding an isolated antigen-specific peptide comprising a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1, or the peptide may have one, two, three or four point mutations (e.g., substitution mutations) compared to SEQ ID NO:1. As mentioned above, such tumor antigen-specific peptides may be, for example, 8 to 35 amino acids in length, or any range that may be derived therein. In some embodiments, the tumor antigen-specific peptide corresponds to a portion of a tumor antigen protein such as VCX1, VCX2, VCX3A, VCX3B or VCY (e.g., VCX3A; GenBank Accession No. AAI26903.1). The term "nucleic acid" is intended to include DNA and RNA, and can be either double-stranded or single-stranded.

[0077] Some embodiments of the present disclosure provide recombinantly produced tumor antigen-specific peptides (e.g., VCX / Y peptides). Thus, the nucleic acid encoding the tumor antigen-specific peptide may be operably linked to an expression vector, and the peptide may be produced in a suitable expression system using methods widely known in the molecular biology arts. The nucleic acid encoding the tumor antigen-specific peptide disclosed herein may be incorporated into any expression vector that ensures good expression of the peptide. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), as long as the vector is suitable for transformation of host cells.

[0078] A recombinant expression vector "suitable for transformation of a host cell" means that the expression vector contains a nucleic acid molecule of the present disclosure and a regulatory sequence that is operably linked to the nucleic acid molecule and that is selected based on the host cell that will be used for expression. The terms "operably linked" or "operably linked" are used interchangeably and are intended to mean that the nucleic acid is linked to a regulatory sequence in a manner that allows for expression of the nucleic acid.

[0079] Thus, the present disclosure provides a recombinant expression vector comprising a nucleic acid encoding a tumor antigen-specific peptide and the necessary regulatory sequences for the transcription and translation of the inserted protein sequence. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal or viral genes (see, for example, the regulatory sequences described in Goeddel (1990)).

[0080] The selection of suitable regulatory sequences generally depends on the host cell selected, and can be easily accomplished by those skilled in the art. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences, including translation initiation signals. In addition, depending on the host cell selected and the vector used, other sequences, such as replication origins, additional DNA restriction sites, enhancers, and sequences that provide inducibility of transcription, may be incorporated into the expression vector. It will also be understood that the necessary regulatory sequences can be provided by the native protein and / or its adjacent regions.

[0081] The recombinant expression vector may also contain a selection marker gene that facilitates the selection of host cells transformed or transfected with the recombinant tumor antigen-specific peptide (e.g., VCX / Y peptide) disclosed herein. Examples of selection marker genes include genes that code for proteins that confer resistance to certain drugs (e.g., G418 and hygromycin), β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. The transcription of the selection marker gene is monitored by changes in the concentration of the selection marker protein (e.g., β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase). If the selection marker gene codes for a protein that confer antibiotic resistance, such as neomycin resistance, transformant cells can be selected with G418. Cells that have incorporated the selection marker gene will survive, while other cells die. This allows the expression of the recombinant expression vector to be visualized and assayed, and in particular, allows the effect of mutations on expression and phenotype to be revealed. It will be understood that a selectable marker can be introduced on a separate vector from the nucleic acid of interest.

[0082] A recombinant expression vector can be introduced into a host cell to produce a transformed host cell. The term "transformed host cell" is intended to include prokaryotic and eukaryotic cells that have been transformed or transfected with a recombinant expression vector of the present disclosure. The terms "transformed by", "transfected by", "transformation" and "transfection" are intended to encompass the introduction of a nucleic acid (e.g., a vector) into a cell by one of many possible techniques known in the art. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the present disclosure may be expressed in bacterial cells (e.g., E. coli), insect cells (using baculovirus), yeast cells or mammalian cells.

[0083] The nucleic acid molecules of the present disclosure may also be chemically synthesized using standard techniques.Various methods of chemically synthesizing polydeoxy-nucleotides are known, including solid-phase synthesis, which is fully automated in commercially available DNA synthesizers, as well as peptide synthesis (see, for example, U.S. Patent Nos. 4,598,049, 4,458,066, 4,401,796 and 4,373,071). III. Antigen-specific cell therapy

[0084] Various embodiments of the present disclosure include the use of antigen-specific cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4 + T cells, CD8 +The present invention relates to administering (if necessary obtained) to a subject antigen-specific T cells (e.g., VCX / Y-specific T cells) to induce, activate and expand functional anti-tumor effector cells. These include: autologous cells (such as tumor infiltrating lymphocytes (TILs)); T cells activated ex vivo using autologous DCs, lymphocytes, artificial antigen presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells isolated by capturing target cell membranes; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and tumor-nonspecific autologous or allogeneic cells that are genetically reprogrammed or "re-ordered" to express tumor-reactive or chimeric TCR molecules that exhibit antibody-like tumor recognition capabilities, known as "T-bodies." These efforts have resulted in numerous protocols for the preparation of T cells and immunization with T cells that can be used in the methods described herein.

[0085] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord and / or lymphoid organs. In some aspects, the cells are human cells. The cells are typically primary cells, such as cells isolated directly from a subject and / or cells isolated and frozen from a subject. Such cells may or may not be isolated from an individual in need of the treatment of the present disclosure. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as a complete T cell population, CD4 + cells, CD8 +The present invention includes cells and subpopulations thereof (e.g., subpopulations defined by function, activation state, maturity, differentiation potential, expansion potential, recirculation potential, localization potential and / or persistence potential, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker profile or cytokine secretion profile, and / or differentiation potential, etc.). The cells may be allogeneic and / or autologous with respect to the subject to be treated. In some aspects, such as for off-the-shelf technology, the cells are pluripotent and / or multipotent, such as stem cells (e.g., induced pluripotent stem cells (iPSCs) and the like). In some embodiments, the method includes isolating the cells from the subject, preparing, processing, culturing and / or manipulating the cells as described herein, and reintroducing the cells into the same patient before or after cryopreservation.

[0086] T cells (e.g., CD4 + and / or CD8 + The subtypes and subpopulations of T cells include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes (e.g., stem cell memory T cells (TSCs) M ), central memory T cells (TC M ), effector memory T cells (T EM ), or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring adaptive regulatory T (Treg) cells, helper T cells (e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells and / or [NRF1] delta / gamma T cells, etc.).

[0087] In some embodiments, one or more of the T cell populations are enriched for or depleted of cells that are positive for or negative for a specific marker (such as, for example, a surface marker). In some cases, such a marker is one that is absent or expressed at a relatively low level in certain populations of T cells (such as, for example, non-memory cells), but is present or expressed at a relatively higher level in certain other populations of T cells (such as, for example, memory cells).

[0088] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on non-T cells (e.g., B cells, monocytes or other leukocytes, etc.), such as, for example, CD14. + or CD8 + The selection process for CD4 + Helper T cells and CD8 + It is used to separate cytotoxic T cells. Such CD4 + Populations and CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers expressed, or expressed to a relatively greater extent, in one or more naive, memory and / or effector T cell subpopulations.

[0089] In some embodiments, CD8 + The T cells are further enriched for or depleted of naive, central memory, effector memory and / or central memory stem cells, such as by positive or negative selection based on one or more surface antigens associated with each subpopulation. In some embodiments, central memory T (T CMEnrichment for such cells is performed to increase efficacy, e.g., to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects are particularly robust in such subpopulations. See Terakura et al. (2012); Wang et al. (2012).

[0090] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from an individual, including a patient, and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, for example, mechanically (by dissociating the tumor, for example, using a gentleMACS™ Dissociator (Miltenyi Biotec, Auburn, Calif.)) or enzymatically (for example, using collagenase or DNase). A single cell suspension of an enzymatic digest of the tumor is cultured in interleukin-2 (IL-2). Cells are cultured at confluence (e.g., about 2×10 6 The cells are cultured for, for example, about 5 to about 21 days, preferably about 10 to about 14 days, until the total number of lymphocytes reaches 100 lymphocytes.

[0091] The cultured T cells can be pooled and rapidly expanded. Rapid expansion results in an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more) over a period of about 10 to about 14 days. More preferably, rapid expansion results in an increase of at least about 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900 or more) over a period of about 10 to about 14 days.

[0092] Expansion can be achieved by any of a number of methods known in the art. For example, T cells can be rapidly expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2) and / or interleukin-15 (IL-15). In some cases, the non-specific T cell receptor stimulation can include an anti-CD3 antibody, such as OKT3 (a mouse monoclonal anti-CD3 antibody, available from Ortho-McNeil®, Raritan, NJ) at around 30 ng / ml. Alternatively, T cells can be rapidly expanded by in vitro stimulation of PBMCs with one or more antigens of the cancer (including antigenic portions thereof; e.g., epitope(s), etc., or cells), which can be expressed from a vector if necessary, in the presence of T cell growth factors, such as 300 IU / ml IL-2 and / or IL-15. In vitro induced T cells are rapidly expanded by restimulation with the same antigen(s) of the cancer pulsed on antigen presenting cells. Alternatively, T cells can be restimulated with, for example, irradiated autologous lymphocytes or with irradiated allogeneic lymphocytes and IL-2.

[0093] Autologous T cells can be modified to express T cell growth factors that promote the growth and activation of autologous T cells. Suitable T cell growth factors include, for example, IL-2, IL-7, IL-15 and / or IL-12. A variety of suitable modification methods are known in the art. See, for example, Sambrook et al. (2001) and Ausubel et al. (1994). In certain aspects, modified autologous T cells express high levels of T cell growth factors. T cell growth factor coding sequences (such as, for example, the coding sequence for IL-12) are readily available in the art, as are a variety of promoters that are operatively linked to T cell growth factor coding sequences to promote high levels of expression. IV. Treatment Method

[0094] Provided herein is a method for treating cancer in an individual or for slowing the progression of cancer in an individual, comprising administering to the individual an effective amount of antigen-specific cell therapy (such as VCX / Y-specific T cell therapy). In a further embodiment, provided is a method for treating cancer, comprising immunizing a subject with a purified tumor antigen or an immunodominant tumor antigen-specific peptide.

[0095] The VCX / Y peptides provided herein can be utilized to develop cancer vaccines or cancer immunogens (e.g., peptide mixes or modified peptide mixes with adjuvants, encoding polynucleotides, and corresponding expression products, such as inactivated virus or other microbial vaccines). These peptide-specific vaccines or immunogens can be used to directly immunize cancer patients to induce anti-tumor immune responses in vivo, or to expand antigen-specific T cells in vitro by stimulating APCs loaded with peptides or encoded polynucleotides. These large numbers of T cells can be adoptively transferred into patients to induce tumor regression.

[0096] Examples of cancers contemplated for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, preneoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer.

[0097] In some embodiments, T cells are autologous. However, cells can be allogeneic. In some embodiments, T cells are isolated from the individual in need of treatment, so that the cells are autologous. If T cells are allogeneic, T cells can be pooled from several donors. Cells are administered to the intended subject in an amount sufficient to suppress, reduce or eliminate the symptoms and signs of the disease being treated.

[0098] In some embodiments, the subject may be administered non-myeloablative lymphodepleting chemotherapy prior to T-cell therapy. Non-myeloablative lymphodepleting chemotherapy may be any suitable such therapy that may be administered by any suitable route. Non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, especially if the cancer is melanoma (which may be metastatic). An exemplary route for administering cyclophosphamide and fludarabine is intravenously. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain aspects, around 60 mg / kg of cyclophosphamide is administered for two days, followed by 25 mg / m 2 Pre- and post-fludarabine is administered over a 5-day period.

[0099] In certain embodiments, a T cell growth factor that promotes the growth and activation of autologous T cells is administered to the subject either simultaneously with the autologous T cells or subsequently. The T cell growth factor can be any suitable growth factor that promotes the growth and activation of autologous T cells. Examples of suitable T cell growth factors include IL-2, IL-7, IL-15 and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2. IL-12 may be used in certain embodiments.

[0100] T cells may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate (single) dose of T cell therapy may be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0101] Intratumoral injection, i.e., injection into the tumor vasculature, is particularly contemplated for isolated, solid, accessible tumors. Local, regional, or systemic administration may also be appropriate. For tumors larger than 4 cm, the volume administered will be about 4 ml to 10 ml (particularly 10 ml). Meanwhile, for tumors smaller than 4 cm, volumes of about 1 ml to 3 ml will be used (particularly 3 ml). Multiple injections delivered as a single dose include volumes of about 0.1 ml to about 0.5 ml. A. Pharmaceutical Compositions

[0102] Also provided herein are pharmaceutical compositions and formulations comprising antigen-specific immune cells (e.g., T cells) and pharmaceutically acceptable carriers. Vaccine compositions for pharmaceutical use in subjects may comprise the tumor antigen peptide (e.g., VCX / Y) compositions disclosed herein and pharmaceutically acceptable carriers. Individuals may receive an effective amount of either or both.

[0103] Pharmaceutical compositions and formulations as described herein can be prepared in the form of lyophilized formulations or aqueous solutions by mixing the active ingredient (e.g., antibody or polypeptide, etc.) having the desired purity with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences, 22nd ed., 2012). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and pharma-ceutically acceptable carriers include, but are not limited to, the following: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives, such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharma- ceutically acceptable carriers herein further include insterstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), such as soluble human PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as, for example, chondroitinases. B. Combination Therapy

[0104] In certain embodiments, the compositions and methods of the present embodiment are accompanied by antigen-specific immune cell population and / or tumor antigen peptide in combination with at least one additional therapy.The additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, or the combination of the above.The additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0105] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or a metastasis inhibitor. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an antiemetic agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy may be one or more chemotherapeutic agents known in the art.

[0106] Immune cell therapy may be administered before, during, after, or in various combinations with additional cancer therapy (such as immune checkpoint therapy). Administration may occur at intervals ranging from simultaneously, to minutes, to days, to weeks. In embodiments in which immune cell therapy is administered to a patient separately from an additional therapeutic agent, it will generally be ensured that a meaningful period does not end after each delivery time, so that the two compounds will still be able to exert a beneficial combined effect on the patient. In such cases, it is contemplated that the patient may be administered antibody therapy and anti-cancer therapy within about 12 hours to 24 hours or 72 hours of each other, more specifically within about 6 hours to 12 hours of each other. In some circumstances, it may be desirable to significantly extend the period for treatment, in which cases days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between each administration.

[0107] Various combinations may be used. In the following example, the antigen-specific immune cell therapy or polypeptide is "A" and the anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0108] Administration of any of the compounds or treatments of the present embodiments to a patient will follow general protocols for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity that may result from the combination therapy. 1.Chemotherapy

[0109] A wide variety of chemotherapeutic agents may be used according to the present embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to imply compounds or compositions administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity in cells, for example, whether and at what stage they affect the cell cycle. Alternatively, agents may be characterized based on whether they can directly crosslink DNA, cause intercalation into DNA, or affect nucleic acid synthesis to induce chromosomal and mitotic abnormalities.

[0110] Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclosphosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridine compounds, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, such as arthrodecitabine, bromodiamine, and bromodiamine; melamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and and cryptophycins; dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictyins; spongistatins; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin hin, phenesterine, prednimustine, trofosfamide and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega 11); dynemicins, such as dynemicin A; bisphosphonates, such as clodronate; esperamicins;Also included are neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, cin), 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (e.g., mitomycin C), mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potofilomycin ( potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane and testolactone; anti-adrenals such as mitotane and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene;Edatraxate; Defofamine; Demecolcine; Diaziquone; Elformithine; Elliptinium acetate; Epothilones; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids, such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pen tostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine ;mitobronitol;mitolactol;pipobroman;gacytosine;arabinoside ("Ara-C");cyclophosphamide;taxoids, such as paclitaxel and docetaxel gemcitabine;6-thioguanine;mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin;vinblastine;platinum;etoposide (VP-16);ifosfamide;mitoxantrone;vincristine;vinorelbine;novantrone;teniposide;edatrexate;daunomycin;a minopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl protein transferase inhibitors, transplatinum, as well as pharmaceutical acceptable salts, acids, or derivatives of any of the above; 2. Radiation therapy

[0111] Other agents that cause DNA damage and are widely used include gamma radiation, commonly known as X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam radiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV radiation. All of these agents most likely affect a wide range of damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome assembly and maintenance. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens over prolonged periods (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the radioisotope, the strength and type of radiation emitted, and uptake by the neoplastic cells. 3. Immunotherapy

[0112] Those skilled in the art will understand that additional immunotherapy can be used in combination with or in conjunction with the method of the present embodiment. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and immune effector molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody alone may serve as the effector of therapy. Alternatively, the antibody may recruit other cells to actually affect cell killing. The antibody may also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte bearing a surface molecule that interacts either directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0113] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Cancer is one of the leading causes of death in the world. Antibody-drug conjugates (ADCs) comprise a monoclonal antibody (MAb) covalently linked to a cell-killing drug. In this approach, the high specificity of the MAb for its antigen target is combined with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells that bear elevated levels of the antigen. The targeted delivery of the drug also minimizes its exposure in normal tissues, thereby resulting in reduced toxicity and improved therapeutic index. The approval of two ADC drugs by the FDA, namely ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, has proven this approach effective. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly relies on the identification and validation of new targets suitable for this endeavor, and the generation of MAbs for targeting. Two criteria for ADC targets are upregulated / high expression levels in tumor cells and unaffected internalization.

[0114] In one aspect of immunotherapy, tumor cells must have some marker that is amenable to targeting, i.e., some marker that is not present on the majority of other cells. There are many tumor markers, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immune stimulating effects. There are also immune stimulating molecules, which include cytokines (e.g., IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, etc.), chemokines (e.g., MIP-1, MCP-1, IL-8, etc.), and growth factors (e.g., FLT3 ligand, etc.).

[0115] Examples of immunotherapies currently under consideration or in use include immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies such as interferon alpha, interferon beta and interferon gamma, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hells et al., 1999); trand et al., 1998); gene therapies, such as TNF, IL-1, IL-2 and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2 and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0116] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints can either enhance or attenuate signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), attenuator of B and T lymphocytes (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), immunoglobulin and mucin domain 3 of T cells (TIM-3), and V-domain Ig suppressor of T-cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0117] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligands or receptors, or in particular antibodies (e.g., human antibodies, etc.) (e.g., International Patent Application Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimeric, humanized or human forms of antibodies. As one skilled in the art would understand, alternative and / or corresponding names may be used for certain antibodies referred to in this disclosure. Such alternative and / or corresponding names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and corresponding names MK-3475 and pembrolizumab.

[0118] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits PDL1 from binding to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits PDL2 from binding to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Publication Nos. 20140294898, 2014022021, and 20110008369, all of which are incorporated by reference herein.

[0119] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in International Publication WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA® and SCH-900475, is an anti-PD-1 antibody described in International Publication WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in International Publication WO2009 / 101611. AMP-224, also known as B7-DCIg, is a soluble receptor of PDL2-Fc fusion described in International Publication WO2010 / 027827 and WO2011 / 066342.

[0120] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. Activation of T cells through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0121] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0122] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Pat. No. 8,119,129, International Publication No. WO 01 / 14424, International Publication No. WO 98 / 42752; the anti-CTLA-4 antibody disclosed in International Publication No. WO 00 / 37504 (CP675,206, also known as tremelimumab, formerly known as ticilimumab); the anti-CTLA-4 antibody disclosed in U.S. Pat. No. 6,207,156; the anti-CTLA-4 antibody disclosed in Hurwitz et al. (1998), Proc Natl Acad Sci USA 95(17):10067-10071; the anti-CTLA-4 antibody disclosed in Camacho et al. (2004), J Clin Oncology 22(145):Abstract no. 2505 (antibody CP-675206); and Mokyr et al. (1998), Cancer Res, 58:5301-5304 can be used in the methods disclosed herein. The teachings of each of the above publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Publication Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114 (all of which are incorporated by reference herein).

[0123] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101 and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., International Publication WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-mentioned antibody. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibody (e.g., at least about 90%, 95% or 99% variable region identity with ipilimumab).

[0124] Other molecules for modulating CTLA-4 include CTLA-4 ligands and CTLA-4 receptors, such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796, and International Patent Application Publications WO1995001994 and WO1998042752, all of which are incorporated herein by reference, and immunoadhesins, such as those described in U.S. Pat. No. 8,329,867, which is incorporated herein by reference. 4.Surgery

[0125] Approximately 60% of people with cancer will undergo some type of surgery, including preventive, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, where all or part of the cancerous tissue is physically removed, excised and / or destroyed, and may be used in combination with other therapies, such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery and microsurgery (Mohs surgery).

[0126] When part or all of cancerous cells, tissues or tumors are removed, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection, or local application to the area with additional anti-cancer therapy. Such treatment may be repeated, for example, every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days or every 7 days, or every week, every 2 weeks, every 3 weeks, every 4 weeks and every 5 weeks, or every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or every 12 months. These treatments may also be of various dosages. 5. Other drugs

[0127] It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. An increase in intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis (such as antibody c225) could be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment. V. Manufactured Articles or Kits

[0128] An article of manufacture or kit is provided that includes the antigen-specific immune cells, TCR, and / or antigenic peptide (e.g., VCX / Y peptide). The article of manufacture or kit may further include a package insert that includes instructions for using the antigen-specific immune cells to treat cancer in an individual, or to slow the progression of cancer in an individual, or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein may be included in the article of manufacture or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin, etc.), or metal alloys (e.g., stainless steel or Hastelloy, etc.). In some embodiments, the container contains the formulation, and a label on or associated with the container may indicate instructions for use. The article of manufacture or kit may further include other buffers, diluents, filters, needles, syringes, and other items desirable from a commercial and user standpoint, including a package insert with instructions for use. In some embodiments, the article of manufacture further comprises one or more additional agents (e.g., chemotherapeutic agents and anti-tumor agents). Suitable containers for such agent(s) include, for example, bottles, vials, bags, and syringes. EXAMPLES

[0129] VI. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques found by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed, and that many changes can still produce the same or similar results without departing from the spirit and scope of the invention. Example 1 - Identification and characterization of tumor antigen-specific peptides

[0130] Screening of overlapping peptide libraries was performed for the recognition target peptides of VCX54 TCR-T cells (Figure 1). 9-mer peptides from an 8 amino acid overlapping peptide library spanning the entire length of the VCX3A protein were screened for targets recognized by VCX54 TCR-T cells. The peptides were pulsed one by one into T2 cells and co-cultured with VCX54 TCR-T. After overnight culture, activated markers of T cells (such as CD137, CD69, IFN-r and TNFα) were detected using intracellular cytokine staining (ICS). It was found that the target peptide VCX54 could induce the response of VCX54 TCR-T. However, it was also found that another peptide VCX118 could induce the response of VCX54 TCR-T cells.

[0131] For VCX118 peptide cross-reactivity detection, different concentrations of VCX118 peptide were used to pulse T2 cells and then co-cultured with VCX54 TCR-T cells. ICS detection indicates that at high concentrations, VCX118 peptide would be cross-recognized by VCX54 TCR-T. The response was greater than that of the first peptide, VCX54. M26 peptide was used as a negative control (Figure 2).

[0132] Different concentrations of VCX118 peptide were pulsed into T2 cells, labeled with calcein-AM, and co-cultured with VCX54 TCR-T cells. Calcein-AM quenching assay was used to detect the killing ability of VCX54 TCR-T in response to antigen. The killing assay showed that at high concentrations, VCX54 TCR-T cells could cross-recognize VCX118 peptide and the response was greater than that of VCX54 peptide (Figure 3).

[0133] VCX118 peptides at different concentrations were pulsed into T2 cells, and HLA-A2 stability analysis was performed to detect the VCX118 peptide binding ability. VCX54 peptide, M26 peptide and M27 peptide were used as controls. From this analysis, VCX118 showed weak binding ability to HLA-A2 (Figure 4).

[0134] [Table 1]

[0135] Peptides of different lengths together with VCX118 were pulsed into T2 cells and co-cultured with VCX54 TCR-T cells. VCX118 peptide was used as a control. ICS data showed that VCX54 TCR-T cells only recognized VCX118 peptide, but not other longer peptides. This indicated that VCX54 TCR-T cells might cross-recognize VCX118 peptide in VCX3A gene. Example 2 - Materials and Methods

[0136] Generation and expansion of VCX118-specific CD8 T cells: Tumor antigen-specific CTLs were generated as previously described (Li, 2005). Leukapheresis PBMCs were stimulated with autologous DCs pulsed with tumor antigen peptides. For induction of dendritic cells, adherent PBMCs were cultured in AIM-V medium (Invitrogen Life Technologies) with GM-CSF and IL-4 for 6 days, after which IL-1β, IL-6, TNF-α and PGE2 were added for maturation. After 1 day, mature DCs were incubated at room temperature for 4 hours with 2×10 cells per ml of 1% human serum albumin (HSA) / PBS in the presence of 3 μg / ml beta-microglobulin. 6 Cells were pulsed with 40 μg / ml of peptide. After washing with 1% HSA / PBS, DCs were cultured at 1.5 × 10 6Cells / ml / well were mixed with PBMCs. IL-21 (30 ng / ml) was added initially and 3-4 days after culture. IL-2 and IL-7 were added 1 day after secondary stimulation to expand activated antigen-specific T cells.

[0137] Six days after the secondary stimulation, cells were stained with VCX / Y peptide / MHC-PE conjugated tetramer and CD8-APC antibody, and then CD8 cells and tetramer-positive cells were selected by ARIA II. The selected VCX / Y-specific CD8 T cells were expanded by the rapid expansion protocol (REP) using PBL and LCL feeder cells under IL-21.

[0138] Peptide-MHC tetramer staining: VCX118-specific CD8 T cells were identified by staining with tetramers of VCX118 peptide / MHC complexes. CD8 T cells were incubated with PE-conjugated tetramers for 20 min, washed, and then stained with APC-conjugated CD8 antibodies for 15 min at room temperature. After washing, cells were analyzed by flow cytometry (LSRFortessa X-20 analyzer).

[0139] To generate T cell clones: Full-length RNA of VCX3A was transfected into mature dendritic cells (DCs). The RNA-transfected DCs were co-cultured with autologous naive T cells at a ratio of DC:T=1:10 in the presence of IL-21. One week later, the RNA-transfected DCs were used to stimulate T cells again. After two rounds of stimulation, CD8+ and tetramer+ double positive T cell populations were selected and expanded by a rapid expansion protocol. T cell clones were obtained by limiting dilution. Highly active CTL clones were screened by tumor cell killing assay.

[0140] 51 Cr release assay: The killing ability of T cell clones or CTL clones to lyse tumor targets was evaluated using standard 51 Cr release assay was used. Tumor or normal cells were treated with 200 μCi 51Cr for 2 hours at 37°C. The labeled target cells were washed and then incubated with effector cells at various ratios in 0.2 ml of complete medium for 4 hours at 37°C. The collected supernatants were counted using an automated gamma counter. Maximal and spontaneous 51 Cr release was determined by incubating labeled target cells in either trypan lysis buffer or medium for 4 hours at 37°C. Each data point was determined as the average of quadruplicate wells. Specific lysis was calculated as follows: % killing = ((specific release - spontaneous release) / (total release - spontaneous release)) x 100.

[0141] IFN-γ release assay: IFN-γ release from T cells was detected by ELISA. T cells were incubated with target cells at a 10:1 ratio in 96-well plates containing 0.2 ml of medium at 37°C. After overnight co-culture, the supernatants were collected and the concentration of IFN-γ was detected using ELISA according to the kit's manual (Invitrogen Life Technologies).

[0142] Intracellular cytokine staining (ICS) assay: T cells were incubated with target cells at a 10:1 ratio overnight at 37°C in the presence of Brefeldin A (BFA). After co-culture, T cells were collected and washed. Cells were first stained with flow antibodies anti-surface markers. After that, cells were washed, fixed with fixation buffer, and then permeabilized using permeabilization solution. Permeabilized cells were then stained with intracellular cytokine flow antibodies. Finally, the levels of cytokine production in cells were analyzed using FACS.

[0143] Statistical analysis: Data analysis was performed using GraphPad Prism (version 6.0e). Normally distributed data were analyzed using parametric tests (Anova or unpaired t-test). Differences in statistical tests were considered significant if the p-value was less than 0.05.

[0144] All of the methods disclosed and claimed herein can be obtained and carried out without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that various changes can be applied to the methods described herein and in the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims. 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Claims

1. An isolated VCX / Y peptide less than or equal to 30 amino acids in length comprising the amino acid sequence of SEVEEPLSQ (SEQ ID NO:1).

2. 2. The peptide of claim 1 further defined as a VCX3A peptide.

3. 2. The peptide of claim 1 which is 25 amino acids or less in length.

4. 4. The peptide of claim 3 which is 20 amino acids or less in length.

5. 5. The peptide of claim 4 which is 15 amino acids or less in length.

6. The peptide of claim 1, consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:

6.

7. A pharmaceutical composition comprising an isolated peptide according to any one of claims 1 to 6 and a pharmaceutical carrier.

8. 8. The composition of claim 7, formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation or subcutaneous injection.

9. The composition of claim 7 or 8, wherein the peptide is contained in a liposome, a lipid-containing nanoparticle, or a lipid-based carrier.

10. The composition according to any one of claims 7 to 9, wherein the pharmaceutical preparation is formulated for injection or inhalation as a nasal spray.

11. An isolated nucleic acid encoding a VCX / Y peptide according to any one of claims 1 to 6.

12. 12. A vector comprising a contiguous sequence comprising the nucleic acid of claim 11 or a contiguous sequence consisting of the nucleic acid of claim 11.

13. A composition for promoting an immune response in a subject, comprising an effective amount of a peptide according to any one of claims 1 to 6.

14. The composition of claim 13 , wherein the subject is diagnosed with cancer.

15. The composition of claim 14, wherein the cancer is thymoma, bladder cancer, uterine cancer, melanoma, sarcoma, cervical cancer or head and neck cancer.

16. The composition according to any one of claims 13 to 15, wherein the subject is a human.

17. The composition of any one of claims 13 to 16, wherein the subject further receives at least a second anti-cancer therapy.

18. 18. The composition of claim 17, wherein the second anti-cancer therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormonal therapy, or surgery.

19. 20. The composition of claim 18, wherein the immunotherapy comprises one or more immune checkpoint inhibitors.

20. 20. The composition of claim 19, wherein the immune checkpoint inhibitor is an anti-PD1 monoclonal antibody.

21. 1. A method for generating VCX / Y-specific immune cells in vitro, comprising: (a) obtaining a starting population of immune cells, if necessary; and (b) contacting a starting population of immune cells with a VCX / Y peptide according to any one of claims 1 to 6, thereby generating VCX / Y-specific immune cells. A method comprising:

22. The method of claim 21, wherein the contacting is further defined as co-culturing the starting population of immune cells with an antigen-presenting cell (APC) that presents a VCX / Y peptide described in any one of claims 1 to 6 on its surface.

23. The method of claim 22, wherein the APC is a dendritic cell.

24. The starting population of immune cells is CD8 ＋ T cells or CD4 ＋ The method of claim 21 , wherein the cell is a T cell.

25. 22. The method of claim 21, wherein the immune cell is a cytotoxic T lymphocyte (CTL).

26. 22. The method of claim 21, wherein obtaining comprises isolating the starting population of immune cells from peripheral blood mononuclear cells (PBMCs).

27. (a) obtaining a starting population of immune cells, if necessary; and (b) contacting a starting population of immune cells with a VCX / Y peptide according to any one of claims 1 to 6, thereby generating VCX / Y-specific immune cells.

28. (a) obtaining a starting population of immune cells, if necessary; and (b) a pharmaceutical composition comprising VCX / Y-specific immune cells produced according to a method comprising contacting a starting population of immune cells with a VCX / Y peptide according to any one of claims 1 to 6, thereby generating VCX / Y-specific immune cells.

29. A composition for treating cancer in a subject, comprising an effective amount of the VCX / Y-specific immune cells described in claim 27.

30. 30. The composition of claim 29, wherein the immune cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), induced pluripotent stem (iPS) cells, or a mixture thereof.

31. 31. The composition of claim 29 or 30, wherein the immune cells are isolated from an umbilical cord.

32. The composition of any one of claims 29 to 31, wherein the immune cells are autologous or allogeneic to the subject.

33. The immune cells are CD8 ＋ T cells, CD4 ＋ 30. The composition of claim 29, which is a T cell or a γδ T cell.

34. The composition of any one of claims 29 to 33, wherein the cancer is thymoma, bladder cancer, uterine cancer, melanoma, sarcoma, cervical cancer or head and neck cancer.

35. The composition of any one of claims 29 to 34, wherein the subject is a human.

36. The composition of any one of claims 29 to 35, wherein the subject has further undergone lymphodepletion prior to administration of the VCX / Y-specific immune cells.

37. 37. The composition of claim 36, wherein lymphodepletion comprises administration of an effective amount of cyclophosphamide and / or fludarabine.

38. The composition of any one of claims 29 to 37, wherein the subject is further receiving at least a second therapeutic agent.

39. 40. The composition of claim 38, wherein the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy, hormone therapy, and / or biological therapy.

40. The composition of claim 39, wherein the VCX / Y-specific immune cells and / or at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesional, percutaneously, subcutaneously, topically, or by direct injection or perfusion.

41. The composition of any one of claims 29 to 40, wherein the subject is determined to have cancer cells that express a protein of the VCX / Y family.

42. 42. The composition of claim 41, wherein the protein is VCX3A.

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