Hormad1 t cell receptors and uses thereof

Abstract

Provided are T cell receptors (TCRs) and TCR variable regions that can selectively bind to Hormad1 peptide / MHC complexes. The TCRs can be used in a variety of treatments, such as autologous Hormad1-TCR adoptive T cell therapy to treat cancer (e.g., solid tumors expressing Hormad1). Methods for expanding populations of relevant T cells are provided.

Description

BACKGROUND

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 930,892, filed November 5, 2019, which is hereby incorporated by reference in its entirety. 1. TECHNICAL FIELD

[0004] The present application relates generally to the fields of immunology and medicine. More particularly, it concerns antigenic peptides and recombinant T cell receptors (TCRs). In some embodiments, the TCRs can be used to treat cancer. 2. BACKGROUND

[0006] Despite the promise of T cell-based therapies to treat a variety of cancers, relapse after administration of immunotherapy or chemotherapy remains an important clinical problem. While aggressive B cell non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL) are generally responsive to a combination of chemotherapy and anti-CD20 monoclonal antibodies (Plosker and Figgitt, 2003), approximately one-third of patients experience repeated relapses and ultimately die from the disease (Chao MP, 2013). Recent studies with chimeric antigen receptor (CAR)-modified T cells targeting CD19 have resulted in a 60% to 90% complete remission (CR) rate in patients with refractory B cell malignancies (Porter et al., 2011; Kochenderfer et al., 2015; Turtle et al., 2016a; Neelapu et al., 2017; Schuster et al., 2015; Turtle et al., 2016b; Locke et al., 2017). In addition, subsets of these patients have experienced long-term remissions, supporting the notion that adoptive T cell therapy can be an effective treatment and can be curative in some patients. Nonetheless, more than half of the patients treated relapse after CD19 CAR T cell therapy, primarily due to loss of CD19 expression on the tumor (Sotillo et al., 2015; Topp et al., 2014; Neelapu et al., 2017). Clearly, new targets for adoptive T cell therapy approaches are needed to further improve clinical outcomes. SUMMARY

[0008] In some aspects, the present disclosure overcomes the limitations of the prior art by providing a Hormad1 peptide (e.g., SEQ ID NO: 5) recognized by HLA-A2 and a T cell receptor (TCR) that can bind to the Hormad1 peptide / MHC I complex. The peptide and TCR can be used, for example, in an adoptive T cell therapy or a soluble T cell therapy to treat cancer.

[0009] One aspect of the present disclosure relates to an isolated Hormad1 peptide of 35 amino acids in length or less, comprising SEQ ID NO: 5, an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5, an amino acid sequence comprising at least 6 contiguous amino acids of SEQ ID NO: 5, or an amino acid sequence comprising only one substitution mutation relative to SEQ ID NO: 5.

[0010] In some embodiments, the peptide comprises an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the peptide comprises an amino acid sequence comprising at least 5, 6, 7, 8, or 9 contiguous amino acids of SEQ ID NO: 5.

[0011] The peptide can be less than 30 amino acids in length, more preferably less than 29 amino acids, more preferably less than 28 amino acids, more preferably less than 27 amino acids, more preferably less than 26 amino acids, more preferably less than 25 amino acids, more preferably less than 24 amino acids, more preferably less than 23 amino acids, more preferably less than 22 amino acids, more preferably less than 21 amino acids, more preferably less than 20 amino acids, less than 19 amino acids, less than 18 amino acids, less than 17 amino acids, less than 16 amino acids, less than 15 amino acids, less than 14 amino acids, less than 13 amino acids, less than 12 amino acids, less than 11 amino acids, or less than 10 amino acids. In some embodiments, the peptide consists of SEQ ID NO: 5. The peptide can be further defined as an immunogenic peptide and / or a peptide capable of inducing a cytotoxic T lymphocyte (CTL) and selectively binding to HLA-A2. The term immunogenic can refer to the generation of an immune response, e.g., a protective immune response. In some embodiments, the peptide is modified. In some embodiments, the modification comprises conjugation to a molecule. The molecule can be an antibody, a lipid, an adjuvant, or a detection moiety (tag).

[0012] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an isolated peptide as described herein or above (e.g., SEQ ID NO: 5) and a pharmaceutical carrier. The pharmaceutical composition can be formulated for parenteral administration, intravenous injection, intramuscular injection, or subcutaneous injection. In some embodiments, the pharmaceutical composition comprises a liposome, a nanoparticle comprising a lipid, or a lipid-based carrier. In some embodiments, the pharmaceutical formulation is formulated for injection. In some embodiments, the pharmaceutical formulation is formulated for inhalation. The pharmaceutical formulation can comprise or consist of a nasal spray.

[0013] Another aspect of the present disclosure relates to an isolated nucleic acid encoding a Hormadl -derived peptide as described herein or above (e.g., SEQ ID NO: 5).

[0014] Another aspect of the present disclosure relates to a vector comprising a nucleic acid described herein or above.

[0015] Also provided are isolated host cells comprising the nucleic acids, peptides, TCRs, and vectors of the present disclosure.

[0016] Another aspect relates to a method of making a cell comprising transferring a nucleic acid or vector of the present disclosure into a cell.

[0017] Another aspect of the present disclosure relates to a method of stimulating an immune response in a mammalian subject comprising administering to the subject an effective amount of a peptide described herein or above (e.g., SEQ ID NO: 5). In some embodiments, the peptide induces, activates, or stimulates proliferation of Hormad1 -specific T cells in the subject. The subject can have a cancer, e.g., breast cancer, lung cancer, bone cancer, endometrial cancer, hematopoietic or lymphoid cancer, gastrointestinal cancer, ovarian cancer, skin cancer, neuroblastoma, testicular cancer, thymoma, bladder cancer, uterine cancer, melanoma, sarcoma, cervical cancer, or head and neck cancer. It is also contemplated that a cancer described herein, e.g., breast cancer, lung cancer, bone cancer, endometrial cancer, hematopoietic or lymphoid cancer, gastrointestinal cancer, ovarian cancer, skin cancer, neuroblastoma, testicular cancer, thymoma, bladder cancer, uterine cancer, melanoma, sarcoma, cervical cancer, or head and neck cancer, can be excluded from the methods of the present disclosure. The cancer can comprise a cancer positive for expression of the peptide. In some embodiments, the subject has been determined to have cells positive for expression or overexpression of the peptide. In some embodiments, the method further comprises administering to the subject an autologous dendritic cell, wherein the peptide is bound to or presented by the autologous dendritic cell. In some embodiments, the peptide and an artificial antigen presenting cell (aAPC) are administered to the subject, wherein the peptide is bound to or presented by the aAPC. In some embodiments, the peptide is operatively linked to an artificial antigen presenting cell (aAPC). The term "operatively linked" refers to situations where two components are combined or are capable of being combined to form a complex. For example, the components can be covalently linked and / or on the same polypeptide, e.g., in a fusion protein, or the components can have a certain degree of binding affinity for each other, e.g., binding affinity resulting from van der Waals forces. In some embodiments, the subject is a human. In some embodiments, the method further comprises administering at least a second anti-cancer therapy. The second anti-cancer therapy can be selected from a chemotherapeutic, a radiation therapy, an immunotherapy, or a surgery.

[0018] Another aspect of the disclosure relates to a method of activating or expanding Hormad1 -specific T cells, comprising: (a) obtaining a starting cell population from a mammalian subject, and preferably from a blood sample of the mammalian subject, wherein the starting cell population comprises T cells; and (b) contacting the starting cell population with a Hormad1 -derived peptide (e.g., SEQ ID NO: 5) as described herein or above ex vivo, thereby activating, expanding Hormad1 -specific T cells in the starting population, and / or stimulating proliferation of Hormad1 -specific T cells. In some embodiments, the contacting is further defined as co-culturing the starting T cell population with an antigen presenting cell (APC), wherein the APC can present the Hormad1 -derived peptide on its surface. In some embodiments, the APC is a dendritic cell. In some embodiments, the dendritic cell is an autologous dendritic cell obtained from the mammalian subject. In some embodiments, the contacting is further defined as co-culturing the starting T cell population with an artificial antigen presenting cell (aAPC). In some embodiments, the artificial antigen presenting cell (aAPC) comprises or consists of poly(lactide-co-glycolide) (PLGA), a K562 cell, paramagnetic beads coated with CD3 and CD28 agonist antibodies, beads or microparticles coupled with HLA-dimers and anti-CD28, or a nano-sized aAPC (nano-aAPC) preferably less than 100 nm in diameter. In some embodiments, the T cells are CD8 + T cells or CD4 + T cells. In some embodiments, the T cells are cytotoxic T lymphocytes (CTLs). In some embodiments, the starting cell population comprises or consists of peripheral blood mononuclear cells (PBMCs). In some embodiments, the method further comprises isolating or purifying the T cells from the peripheral blood mononuclear cells (PBMCs). In some embodiments, the mammalian subject is a human. The method can further comprise reinfusing or administering the activated or expanded Hormad1 -specific T cells to the subject.

[0019] Another aspect of the disclosure relates to Hormad1 -specific T cells activated or expanded according to the methods herein or above.

[0020] Another aspect of the disclosure relates to a pharmaceutical composition comprising Hormad1 -specific T cells activated or expanded according to the methods herein or above.

[0021] Another aspect of the present disclosure relates to an engineered T cell receptor (TCR) having antigenic specificity for Hormadl or SEQ ID NO: 5, wherein the TCR comprises an amino acid sequence of SEQ ID NOs: 6, 7, 8, 9, 10, and / or 11. The engineered TCR can comprise a TCRa CDR3 comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 and a TCRP CDR3 comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11. The engineered TCR can comprise a TCRa CDR3 comprising an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 8 and a TCRP CDR3 comprising an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the TCR comprises a TCRa CDR1 and / or CDR2 comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 6 and / or 7, respectively, and a TCRP CDR1 and / or CDR2 comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 9 and / or 10, respectively.In some embodiments, the TCR comprises a TCRa CDR1 and / or CDR2 comprising an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 6 and / or 7, respectively, and a TCRP CDR1 and / or CDR2 comprising an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 9 and / or 10, respectively. In some embodiments, the engineered TCR comprises: (i) an a chain variable region having the amino acid sequence of SEQ ID NO: 13 or 2, or a sequence having at least 90% sequence identity to SEQ ID NO: 13 or 2; and / or (ii) a β chain variable region having the amino acid sequence of SEQ ID NO: 15 or 4, or a sequence having at least 90% sequence identity to SEQ ID NO: 15 or 4. The engineered TCR can bind to SEQ ID NO: 5 when bound to HLA-A2. The engineered TCR can bind to an MHC / peptide complex of SEQ ID NO: 5 bound to HLA-A2. In some embodiments, the TCR comprises an a chain variable region having at least 95% identity to the amino acid sequence of SEQ ID NO: 13 or 2, and / or a β chain variable region having at least 95% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the TCR comprises an a chain variable region having at least 99% identity to the amino acid sequence of SEQ ID NO: 13 or 2, and / or a β chain variable region having at least 95% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the TCR comprises an a chain variable region having at least 95% identity to the amino acid sequence of SEQ ID NO: 13 or 2, and / or a β chain having at least 99% identity to the amino acid sequence of SEQ ID NO: 15 or 4. In some embodiments, the TCR comprises an a chain variable region of SEQ ID NO: 13 or 2, and a β chain of SEQ ID NO: 15 or 4.In some embodiments, the soluble TCR is further defined as a single-chain TCR (scTCR), in which the a chain and the β chain are covalently linked by a flexible linker. In some embodiments, the TCR comprises or consists of a bi-specific TCR. The bi-specific TCR can comprise a scFv that targets or selectively binds CD3.

[0022] Another aspect of the disclosure relates to a multivalent TCR complex comprising a plurality of TCRs as described herein or above. In some embodiments, the multivalent TCR comprises 2, 3, 4, or more TCRs associated with one another. In some embodiments, the multivalent TCR is present in a lipid bilayer, a liposome, or attached to a nanoparticle. In some embodiments, the TCRs are associated with one another by a linker molecule or a non-naturally occurring disulfide bond.

[0023] Another aspect of the disclosure relates to a nucleic acid comprising or consisting of a nucleotide sequence encoding a TCR as described herein or above. In some embodiments, the nucleic acid comprises a cDNA encoding a TCR.

[0024] Another aspect of the disclosure relates to an expression vector comprising the nucleic acid described above. The vector can comprise both a TCR a and a TCR β gene on the same nucleic acid. In some embodiments, the nucleotide sequence encoding the TCR is under the control of a promoter. In some embodiments, the expression vector is a viral vector (e.g., a retroviral vector or a lentiviral vector).

[0025] Another aspect of the disclosure relates to a host cell engineered to express a TCR as described herein or above, preferably wherein the host cell comprises an expression vector as described herein or above. In some embodiments, the cell is a T cell, an NK cell, an invariant NK cell, an NKT cell, a mesenchymal stem cell (MSC), or an induced pluripotent stem (iPS) cell. In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is isolated from umbilical cord. In some embodiments, the T cell is a CD8+ T cell, a CD4+ T cell, or a γδ T cell. In some embodiments, the T cell is a regulatory T cell (Treg). In some embodiments, the cell is autologous. In some embodiments, the cell is allogeneic.

[0026] Another aspect of the present disclosure relates to a method for engineering a host cell as described above, comprising contacting an immune cell with a nucleic acid as described herein or above or an expression vector as described herein or above. In some embodiments, the immune cell is a T cell or a peripheral blood lymphocyte. In some embodiments, the contacting is further defined as transfection or transduction. The transfection can comprise electroporating RNA encoding a TCR as described herein or above into the immune cell. The method can further comprise producing a viral supernatant from the expression vector described herein or above to transduce the immune cell. In some embodiments, the immune cell is a stimulated lymphocyte (e.g., a human lymphocyte). In some embodiments, the stimulating comprises contacting the immune cell with OKT3 and / or IL-2 or incubating the immune cell in OKT3 and / or IL-2. In some embodiments, the method further comprises sorting the immune cell to isolate TCR engineered T cells. The method can further comprise T cell cloning by serial dilution. In some embodiments, the method further comprises expanding the T cell clones by a rapid expansion protocol.

[0027] Another aspect of the present disclosure relates to a method of treating a cancer in a mammalian subject, comprising administering to the subject an effective amount of a TCR engineered cell as described herein or above, wherein the cancer expresses Hormadl. In some embodiments, the TCR engineered cell is a T cell or a peripheral blood lymphocyte. In some embodiments, the T cell is a CD8+ T cell, a CD4+ T cell, or a Treg. In some embodiments, the cancer is a breast cancer, a lung cancer, an esophageal cancer (esophagus cancer), a bone cancer, an endometrial cancer, a hematopoietic cancer or a lymphoid cancer, a gastrointestinal cancer, an ovarian cancer, a skin cancer, a neuroblastoma, a testicular cancer, a thymoma, a bladder cancer, a uterine cancer, a melanoma, a sarcoma, a cervical cancer, a head and neck cancer. In some embodiments, the cancer is a solid tumor. The subject can be a human. In some embodiments, the TCR engineered cell is autologous or allogeneic to the subject. The method can further comprise lymphodepleting the subject prior to administering the Hormadl specific T cells. In some embodiments, the lymphodepletion comprises administering cyclophosphamide and / or fludarabine. The method can further comprise administering a second anti-cancer therapy to the subject. In some embodiments, the second therapy is a chemotherapy, an immunotherapy, a surgery, a radiation therapy, or a biological therapy. In some embodiments, the TCR engineered cell and / or at least the second therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally, or by direct injection or perfusion. In some embodiments, the subject is determined to have or diagnosed with a cancer cell that overexpresses Hormadl.

[0028] In some aspects, methods for treating cancer (e.g., breast cancer, lung cancer, etc.) are provided that include immunizing a subject with a purified tumor antigen or immunodominant tumor antigen specific peptide, such as a Hormadl peptide (SEQ ID NO: 5). In some embodiments, the peptide can be injected into a solution (e.g., a saline solution) as a vaccine or to elicit an immune response against the peptide. For example, to enhance solubility of the peptide and / or to increase an immune response in a subject, an adjuvant can be included in the formulation or solution (e.g., Massarelli et al. 2019). In some embodiments, peptide-pulsed mature dendritic cells can be administered to a subject. Methods that can be used to elicit an immune response or anti-cancer response against the peptide in a subject include, for example, Wen et al. (2019) and Massarelli et al. (2019). In some embodiments, the Hormadl peptide (SEQ ID NO: 5) is bound to or presented by autologous dendritic cells that can be reinfused into a subject or human patient.

[0029] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the field of cell and molecular biology to indicate that a value includes the standard deviation of the error in the apparatus or method used to determine the value.

[0030] The use of the singular herein will be understood to include the plural unless the context otherwise requires. What is considered to be the plural is indicated by the use of the term “at least one,” which means one or more than one.

[0031] The terms “or” and “and / or” are used herein to describe multiple components that are combined or that can be combined with each other, or to describe multiple components that are either disjunctively or conjunctively related. For example, “x, y, and / or z” can mean “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 can be specifically excluded from an embodiment.

[0032] The words “comprise,” “have,” “include,” “contain,” “characterized by,” “characterized in that,” or “contain,” and any variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0033] The compositions and methods described herein may, depending on their intended use, "comprise / include" any ingredient or step disclosed throughout this specification, "consistently constitute" or "compose of". The phrase "consistently constitute" excludes any unspecified element, step, or ingredient. The phrase "consistently constitute" limits the scope of the subject matter to the specified substance or step and those substances or steps that do not substantially affect their essential and novel characteristics. Some embodiments described in the context of the term "comprise / include" are contemplated for implementation in the context of the terms "consistently constitute" or "consistently constitute".

[0034] It is specifically contemplated that any limitations discussed with respect to one embodiment of the invention may be applied to any other embodiment of the invention. Furthermore, any composition of the invention can be used in any method of the invention, and any composition of the invention can be produced or utilized using any method of the invention. Some aspects of the embodiments set forth in the examples are also embodiments that may be practiced in the context of some embodiments discussed elsewhere in different embodiments or elsewhere in this application (e.g., in the summary of the invention, detailed description of the invention, claims, and description of the drawings).

[0035] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples indicate some particular embodiments of the invention, they are given by way of example only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description. Attached Figure Description

[0037] This patent or application document contains at least one color-drawn drawing. Upon request and payment of the necessary fees, the official authority will provide a copy of the patent or application publication with the color drawing.

[0038] The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these drawings in conjunction with the detailed description of specific embodiments given herein.

[0039] FIGS. 1A-1D Hormad1 expression in normal and tumor tissues. FIG. 1A Hormad1 expression in normal tissues. FIG. 1B High Hormad1 expression is observed in esophageal cancer, lung cancer, and head and neck cancer. FIG. 1C High Hormad1 expression is observed in cervical cancer, bladder cancer, and acute myeloid carcinoma. FIG. 1D High Hormad1 expression in melanoma and gastric cancer.

[0040] FIG. 2 T cell receptor (TCR) repertoire analysis of Hormad1-56 A12 CTL cell line. TCR a and β chains were cloned from Hormad1-56 A12 CTL using 5'-RACE PCR. Both a and β chains were sequenced and annotated using the IMGT / V-QUEST tool. The TCR usage and CDR3 sequences of a and β chains are shown.

[0041] FIG. 3 Hormad1-56 antigen-specific T cell receptor engineered T cell (TCR-T) generation. Full-length TCR a and β chains were inserted into retroviral vector pMSGV3 and then used to infect peripheral blood mononuclear cells (PBMCs) using recombinant retroviral vectors. Empty retroviral vector was used as a control. After infection, CD8+ / tetramer+population was observed by flow cytometry (FCM) detection. After tetramer-guided sorting and expansion, high purity TCR-T cells were generated.

[0042] FIGS. 4A-4F: Hormad1-56 TCR-T cell killing assays with different targets. (FIG. 4A) Peptide titration assay: T2 cells were pulsed with different concentrations of Hormad1-56 peptide as targets. The effector to target (E:T) ratio was 20:1. (FIGS. 4B-F) Tumor target killing assays: Hormad1-56 TCR-T cells were co-cultured with: (FIG. 4B) tumor cell lines H1395 (HLA-A2+, Hormad1+) and H522 (HLA-A2+, Hormad1-); (FIG. 4C) tumor cell lines H1299 (HLA-A2-, Hormad1+) and H1299-A2 (HLA-A2 forced expressing, Hormad1+); (FIG. 4D) tumor cell lines H1355 (HLA-A2+, Hormad1+) and H1755 (HLA-A2+, Hormad1-); (FIG. 4E) K562-A2 cell lines forced expressing eGFP control gene or Hormad1 gene; or (FIG. 4F) H522 tumor cell lines forced expressing eGFP control gene or Hormad1 gene. For tumor target killing assays, the effector to target (E:T) ratio was 40:1 to 1.25:1. The ability of Hormad1-56 TCR-T to lyse different targets was detected by Cr51 release assay (CRA).

[0043] FIG. 5: Functional detection of Hormadl-56 TCR-T cells by intracellular cytokine staining (ICS) assay. Hormadl-56 TCR-T cells were co-cultured with H522, H1395, H1755, H1355, DFC1032, HSAEC2-KT, H1299, H1299-A2, H522-eGFP, H522-Hormadl, K562-A2-eGFP, K562-A2-Hormadl at a ratio of E:T = 10: 1. After overnight co-culture, the markers of TCR pathway downstream activation, CD137, CD69, IFN-g and TNF-a were detected by ICS assay. The levels of CD137, CD69, IFN-g and TNF-a of Hormadl-56 TCR-T cells were significantly enhanced when co-cultured with positive targets H1395, H1355, H1299-A2, H522-Hormadl, K562-A2-Hormadl compared to negative controls.

[0044] FIGS. 6A-6B : Full sequence of Hormadl-TCR. FIG. 6A ) Full sequence of Hormadl CTL A12 TCR (TRAV4*01F, TRBV13*01F) alpha chain. (SEQ ID NO: 2) FIG. 6B ) Full sequence of Hormadl CTL A12 TCR (TRAV4*01F, TRBV13*01F) beta chain. (SEQ ID NO: 4) Blue: signal peptide; yellow: variable region; red: CDR1, CDR2, CDR3; black: constant region. DETAILED DESCRIPTION

[0046] In some aspects, peptides derived from Hormadl recognized by MHC I (HLA-A2) are provided and can be used in methods for treating cancer. For example, the HLA-A2 restricted T cell epitope YLDDLCVKI (SEQ ID NO: 5) can be used to expand or activate antigen-specific T cells in vitro. The expanded or activated antigen-specific T cells can be used in cancer treatment, such as adoptive cell transfer therapy. Thus a variety of cancers expressing Hormadl, such as lung cancer, cervical cancer, esophageal cancer, head and neck cancer, leukemia or solid tumor, can be treated in a mammalian subject (e.g., a human).

[0047] In other aspects, cloned T-cell receptor (TCR) sequences (e.g., SEQ ID NO: 1 to 4) capable of binding to the Hormad1-derived peptide / HLA-A2 complex are provided. The TCRs of this disclosure can be used to generate T cells that recognize the Hormad1-derived peptide / HLA-A2 complex. Such T cells include engineered T cells expressing the TCR (TCR-T). Those engineered T cells can be used to treat cancer. Related soluble TCRs (sTCRs) and single-chain TCRs (scTCRs) are also provided and can also be used to generate engineered T cells for adoptive cell transfer therapy to treat cancer.

[0048] The provided peptide and TCR, or the antigen-binding domain or functional fragment of the TCR, may be included in a variety of other constructs. For example, in some embodiments, the antigen-binding domain of the TCR may be included in a chimeric antigen receptor (CAR). The peptide (e.g., SEQ ID NO:5) may also be used to generate MHC-peptide multimers or tetramers (e.g., HLA-A2 / peptide tetramers), and the peptide may be included in the immunogenic composition.

[0049] I. Engineered T-cell receptors

[0050] In several aspects, T-cell receptors (TCRs) that specifically bind to the Hormad1-derived peptide (e.g., SEQ ID NO:5) / MHC I (HLA-A2) complex are provided. Therefore, these TCRs can be used to target T cells to cancer cells expressing the Hormad1 protein. The antigen-binding region of the TCR (e.g., FIGS. 6A-6B The CDR1, CDR2, and CDR3 shown may be contained within a soluble TCR (sTCR) or a chimeric antigen receptor (CAR) as an extracellular domain containing an antigen-binding region. In some aspects, the TCR is an isolated or purified TCR. The polynucleotide encoding the TCR can be transfected into cells (e.g., autologous or allogeneic cells) that can be used for adoptive cell transfer therapy (also known as “adoptive cell therapy”).

[0051] In some implementations, the host cell, such as T cells as described in this disclosure (e.g., CD4), + T cells, CD8 +T cells, αβ T cells, γδ T cells, and Tregs), NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), or induced pluripotent stem (iPS) cells can be genetically engineered to express receptors, such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, autologous or allogeneic cells (e.g., isolated from umbilical cords or healthy donors) are modified to express a T cell receptor (TCR) with antigenic specificity for a short peptide derived from a cancer antigen (e.g., Hormad1 and SEQ ID NO: 5), such as when presented in the context of a particular MHC allele (e.g., HLA-A2). In some embodiments, the TCR has antigenic specificity for a Hormad1 -derived peptide (SEQ ID NO: 5) / HLA-A2 complex. In some embodiments, the engineered TCR comprises the CDR1, CDR2, and CDR3 regions of the TCRa and TCRP chains as shown in FIGS. 6A-6B In some embodiments, the engineered TCR has an a chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 2 and / or a P chain comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the TCR has an a chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 and / or a P chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 3. Suitable methods for making modifications to amino acid sequences (e.g., to introduce substitution, deletion, or insertion mutations) are known in the art.

[0052] A. T cell receptors (TCRs)

[0053] In some aspects, provided herein are recombinant T cell receptors (TCRs). A "T cell receptor" or "TCR" generally includes a variable alpha and beta chain (also referred to as TCRa and TCRP, respectively) or a variable gamma and delta chain (also referred to as TCRy and TCR5, respectively), and is capable of specifically binding an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is in the alpha beta form and is referred to as TCRaP. In certain embodiments, the engineered TCR has an alpha chain variable region of SEQ ID NO: 2 and / or a beta chain variable region of SEQ ID NO: 4. In some embodiments, the TCR alpha chain is encoded by a nucleic acid comprising or consisting of SEQ ID NO: 1, and the beta chain is encoded by a nucleic acid comprising or consisting of SEQ ID NO: 3, respectively.

[0054] Some embodiments of the disclosure relate to engineered T cell receptors. The term "engineered" means that the T cell receptor has a TCR variable region grafted onto a TCR constant region to make a chimeric polypeptide that binds to the peptide and antigen of the disclosure. In certain embodiments, the TCR comprises intervening sequences for cloning, detection, enhancing expression of the construct, or for a therapeutic control of the construct, but not present in an endogenous TCR, such as a multiple cloning site, a linker, a hinge sequence, a modified hinge sequence, a modified transmembrane sequence, a detection polypeptide or molecule, or a therapeutic control that can allow selection or screening of cells comprising the TCR.

[0055] In some embodiments, the TCR comprises non-TCR sequences. Thus, certain embodiments relate to TCRs having sequences that are not from TCR genes. In some embodiments, the TCR is chimeric because it comprises sequences from at least two TCR genes that are not necessarily found together in nature, in addition to sequences normally found in TCR genes.

[0056] The following provided TCR has been determined herein to selectively bind to a Hormadl -derived peptide (e.g., SEQ ID NO: 5) / HLA-A2 complex:

[0057] Alpha chain DNA sequence (SEQ ID NO: 1)

[0058]

[0059] Alpha chain protein sequence (SEQ ID NO: 2):

[0060]

[0061] Beta chain DNA sequence (SEQ ID NO: 3):

[0062]

[0063] Beta chain protein sequence (SEQ ID NO: 4):

[0064]

[0065] HLA-A2 restricted peptide derived from Hormad1 (SEQ ID NO: 5): YLDDLCVKI

[0066] Alpha chain CDR1 peptide (SEQ ID NO: 6): NIATNDY

[0067] Alpha chain CDR2 peptide (SEQ ID NO: 7): GYKTK

[0068] Alpha chain CDR3 peptide (SEQ ID NO: 8): LVGARGTALIF

[0069] Beta chain CDR1 peptide (SEQ ID NO: 9): PRHDT

[0070] Beta chain CDR2 peptide (SEQ ID NO: 10): FYEKMQ

[0071] Beta chain CDR3 peptide (SEQ ID NO: 11): ASSPTGQGSYEQY

[0072] Alpha chain variable region DNA sequence (SEQ ID NO: 12):

[0073]

[0074] Alpha chain variable region protein sequence (SEQ ID NO: 13):

[0075]

[0076] Beta chain variable region DNA sequence (SEQ ID NO: 14):

[0077]

[0078] Beta chain variable region protein sequence (SEQ ID NO: 15):

[0079]

[0080] The term "TCR" should be understood to encompass both full-length native TCR polypeptides, as well as functional fragments thereof in various combinations, including alpha beta forms or gamma delta forms, unless otherwise indicated. A "functional" TCR or fragment thereof used herein is capable of binding its cognate subunit (e.g., alpha to beta or gamma to delta) to form a full-length TCR or a truncated TCR that is still capable of binding its cognate peptide presented in the context of an appropriate MHC allele (e.g., HLA-A2).

[0081] Accordingly, for purposes herein, reference to a TCR includes any TCR or TCR fragment that can bind an antigenic peptide, such as a TCR antigen binding portion that binds a particular antigenic peptide bound in an MHC molecule (i.e., an MHC-peptide complex). The terms "antigen binding portion" or "antigen binding fragment" of a TCR, used interchangeably herein, refer to a molecule comprising a portion of a TCR that binds an antigen (e.g., an MHC-peptide complex) that is bound by a full TCR.

[0082] The variable domains of TCR chains are generally understood to form loops or complementarity determining regions (CDRs), analogous to those present in immunoglobulins that confer antigen recognition; in TCRs, the CDRs determine peptide specificity by forming the binding site of the TCR molecule. Generally, as with immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; see also Lefranc et al., 2003). The CDR3 region on both the alpha and beta chains of a TCR is generally understood to be involved in binding the processed antigenic peptide. In some embodiments, the variable region of the beta chain can comprise an additional hypervariable (HV4) region.

[0083] Alpha / beta and gamma / delta TCRs are structurally similar, but the T cells expressing them can have different anatomical locations or functions. As will be appreciated by one of skill in the applicable art, a TCR is present on the surface of a T cell (or T lymphocyte), where it can recognize an antigen-derived peptide bound to a major histocompatibility complex (MHC) molecule. A TCR comprises different regions, including: a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al, Immunobiology: The Immune System in Health and Disease, 3 rdEd., Current Biology Publications, p. 433, 1997). The TCR a and β chains can associate with constant proteins of the CD3 complex that are involved in mediating signal transduction.

[0084] In some embodiments, the TCR comprises a functional fragment of a Hormadl-TCR. In some embodiments, the functional fragment comprises a constant domain and a variable domain of a Hormadl-TCR. Similar to immunoglobulins, the extracellular portion of a TCR chain (e.g., a chain, β chain) can comprise two immunoglobulin domains, a variable domain (e.g., V a ; typically amino acids 1-116 based on Kabat numbering, Kabat et al., “Sequences of Proteins of Immunological Interest,” US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.) and one constant domain adjacent to the cell membrane (e.g., an a chain constant domain or C a , typically amino acids 117-259 based on Kabat, a β chain constant domain, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of a TCR formed by two chains (e.g., a β form or γδ form) comprises two membrane proximal constant domains and two membrane distal variable domains (which contain CDRs). The constant domains of the TCR domains comprise short connecting sequences, with cysteine residues forming disulfide bonds, such that a linkage is established between the two chains. In some embodiments, the formation of additional interchain disulfide bonds can be facilitated by the addition of a single cysteine on each receptor chain to improve TCR gene transfer, e.g., as described in Cohen et al. (2007).

[0085] A CDR can also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 18, 19, 20, 21, 22, 23, or more contiguous amino acid residues (or any range derivable therein) flanking one or both sides of a particular CDR sequence in the context of the variable region of a TCR-a or TCR-b polypeptide; thus, one or more additional amino acids can be present at the N- or C-terminus of a particular CDR sequence (such as those shown in the variable regions of SEQ ID NOs: 13 and 15). Alternatively, or in combination, a CDR can also be a fragment of a CDR described herein and can lack at least 1, 2, 3, 4, or 5 amino acids from the C- or N-terminal end of a particular CDR sequence.

[0086] In some embodiments, the TCR chains each comprise a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chains comprise a cytoplasmic tail. In some cases, the TCR can associate with other molecules, such as CD3. For example, a TCR comprising a constant domain and a transmembrane domain can anchor the protein in the cell membrane and enable it to associate with the invariant subunits of the CD3 signaling apparatus or complex.

[0087] CD3 is a multi-subunit complex comprising different chains: gamma, delta, epsilon, and zeta chains. For example, in mammals, the complex can comprise a CD3y chain, a CD35 chain, two CD3e chains, and a homodimer of CD3z chains. The CD3y, CD35, and CD3e chains are highly related cell surface proteins of the immunoglobulin superfamily. The transmembrane domains of the CD3y, CD35, and CD3e chains are negatively charged, which is a feature that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3y, CD35, CD3e, and CD3z chains each comprise a conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM). An ITAM is a repeatable, conserved amino acid sequence and is involved in the signaling capability or signal transduction of the TCR complex. These accessory molecules have negatively charged transmembrane domains and play a role in transmitting signals from the TCR into the cell. The CD3 chains and zeta chain form, along with the TCR, a so-called T cell receptor complex (TCR complex).

[0088] In some embodiments, the TCR comprises a heterodimer comprising one TCRa polypeptide and one TCRb polypeptide. The TCR can comprise a heterodimer comprising one TCRy polypeptide and one TCR5 polypeptide. In some embodiments, the TCR comprises a single chain TCR (scTCR). In some embodiments, the polypeptides of the TCR heterodimer are covalently linked. In some embodiments, the covalent linkage is through one or more disulfide bonds. In some embodiments, the one or more disulfide bonds comprise naturally occurring disulfide bonds as found in native TCRs. In some embodiments, the one or more disulfide bonds comprise non-naturally occurring disulfide bonds not found in native TCRs.

[0089] The TCRs of the present disclosure can be expressed in cells, e.g., T cells, by transfecting the cells with nucleic acids encoding the TCR using a variety of methods as will be appreciated by those skilled in the art. For example, viral vectors can be used to transfect T cells (e.g., Levine et al., 2017). In some embodiments, non-viral methods are used to transfect T cells (e.g., as described in Riet et al., 2013), including electroporation methods (e.g., Zhang et al., 2018).

[0090] B. Soluble TCRs

[0091] In some embodiments, the present disclosure provides soluble TCRs, which can include TCR variable regions specific for the Hormadl -derived peptides provided herein (e.g., SEQ ID NOs: 13 and 15). Soluble TCRs are useful not only for research purposes to study specific TCR-MHC interactions, but also are useful in potentially being used as diagnostic tools to detect infection or to detect autoimmune disease biomarkers. Soluble TCRs also find application in staining, e.g., for staining cells for the presence of specific peptide antigens presented in the context of MHC. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting specific antigens. Soluble TCRs can also be used to inhibit T cells, e.g., those reactive with autoimmune peptide antigens.

[0092] In the context of the present application, “solubility” is defined as the ability of a TCR to be purified as a monodisperse heterodimer in phosphate buffered saline (PBS) (KCI 2.7 mM, KH2PO4 1.5 mM, NaCI 137 mM, and Na2PO4 8 mM, pH 7.1 to 7.5. Life Technologies, Gibco BRL) at a concentration of 1 mg / ml and more than 90% of the TCR remains as a monodisperse heterodimer after incubation at 25°C for 1 hour.

[0093] In some aspects, the present disclosure provides soluble T cell receptors (sTCRs) comprising (i) all or a portion of a TCR a chain (e.g., SEQ ID NO: 1 or 2), except for its transmembrane domain, and (ii) all or a portion of a TCR β chain (e.g., SEQ ID NO: 3 or 4), except for its transmembrane domain, wherein each of (i) and (ii) comprises a functional variable domain and at least a portion of a constant domain of a TCR chain and are linked by an inter-constant domain residue disulfide bond that is not present in native TCRs. In some aspects, the soluble TCR comprises a TCR a or γ chain extracellular domain that is dimerized to a TCR β or δ chain extracellular domain, respectively, by a pair of C-terminal dimerization peptides (e.g., leucine zippers) (International Patent Publication No. WO 99 / 60120; U.S. Patent No. 7,666,604).

[0094] In some embodiments, the entire antigen binding region comprising TCR variable regions (e.g., see FIGS. 6A-6B ) can be included in the sTCR. The sTCR can be a single chain T cell receptor (scTCR), in which the variable regions from the a and β chains (Vα and Vβ) are covalently linked by a flexible linker, and the ends of the variable regions (typically the ends of Vβ that are not linked to the linker) are covalently linked to a therapeutic compound (e.g., a toxin, a chemotherapeutic agent, etc.) or an imaging agent. The sTCR can recognize an intracellular or extracellular epitope when presented by an MHC, and the sTCR can be used to identify natural peptide ligands in disease (e.g., Walseng et al., 2015; Boulter et al., 2005). Thus, the sTCR can be administered to a subject, e.g., a human patient, to visualize tumor cells or to deliver a therapeutic compound to cancer cells to treat cancer. A variety of therapeutic molecules or toxins can be delivered to a cell, e.g., a cancer cell expressing a Hormad1 -derived peptide / HLA-A2 complex, by the sTCR, e.g., a maytansinoid, a calicheamicin, a STING agonist, a cytokine, a chemokine, a costimulatory agonist (e.g., OX40), or other chemotherapeutic agent. In this way, the sTCR can be used to target delivery of a therapeutic molecule to a tumor site. In some embodiments, the sTCR comprises or is covalently linked to a fluorescent or radioactive probe. 131 I, an auristatin, a maytansinoid, a calicheamicin, a STING agonist, a cytokine, a chemokine, a costimulatory agonist (e.g., OX40), or other chemotherapeutic agent. In this way, the sTCR can be used to target delivery of a therapeutic molecule to a tumor site. In some embodiments, the sTCR comprises or is covalently linked to a fluorescent or radioactive probe.

[0095] The soluble TCRs of the present disclosure (which can be human or produced in human cells) can be provided in substantially pure form or as a purified or isolated preparation. For example, it can be provided in a form that is substantially free of other proteins.

[0096] The plurality of soluble TCRs of the present disclosure can be provided in a multivalent complex. Thus, in one aspect, the present disclosure provides a multivalent T cell receptor (TCR) complex comprising a plurality of soluble T cell receptors as described herein. Each of the plurality of soluble TCRs is preferably identical. The multivalent TCR can comprise two or more ligand-binding TCR a / b subunits (see, e.g., Schamel et al., 2005).

[0097] The multivalent TCR complex typically comprises a multimer of two or three or four or more T cell receptor molecules associated with each other (e.g., covalently or otherwise linked to each other), preferably through a linker molecule. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin, and extravidin, each of which has four binding sites for biotin. Thus, biotinylated TCR molecules can be formed into a T cell receptor multimer having multiple TCR binding sites. The number of TCR molecules in the multimer will depend on the amount of TCR relative to the amount of linker molecule used to make the multimer, and also on whether any other biotinylated molecules are present. Preferred multimers are dimeric, trimeric, or tetrameric TCR complexes.

[0098] The TCR or multivalent TCR complex can be attached to a membrane structure (e.g., a liposome) or a solid structure, which is preferably a particle, such as a bead (e.g., a latex bead). In some embodiments, the structure is coated with the T cell receptor multimer rather than individual T cell receptor molecules. In the case of a liposome, the T cell receptor molecules or multimers thereof can be attached to or otherwise associated with the membrane. Techniques for this are well known to those of skill in the art.

[0099] Labels or other moieties, such as toxic or therapeutic moieties, can be included in the multivalent TCR complex. For example, labels or other moieties can be included in the mixed molecular multimers. One example of such a multimeric molecule is a tetramer comprising three TCR molecules and one peroxidase molecule. This can be achieved by mixing the TCR and the enzyme in a molar ratio of about 3: 1 to produce tetrameric complexes, and isolating the desired complex relative to any complexes that do not comprise the correct ratio of molecules. These mixed molecules can comprise any combination of molecules, provided that steric hindrance does not impair or significantly impair the desired function of the molecules. Since steric hindrance is less likely to occur, the positioning of the binding sites on the streptavidin molecule can be adapted for the mixed tetramers.

[0100] In some embodiments, the peptides provided herein (e.g., SEQ ID NO: 5) can be used to generate MHC-peptide tetramers (e.g., HLA-A2 / peptide tetramers). These tetramers can be used to isolate epitope-specific T cells (e.g., tumor infiltrating lymphocytes or TILs) from patient samples or in vitro following pulsing of professional APCs with a particular Hormadl peptide, Hormadl protein, or nucleotide sequence encoding a particular Hormadl peptide or Hormadl protein. In some cases, MHC-peptide tetramers can be used to visualize T cells in tissues (e.g., Dileepan et al., 2015). MHC multimer-guided approaches can also be used to facilitate isolation of functional T cell receptors from single cells that can be used for immunotherapy. For example, direct isolation of pairs of full-length TCR sequences from non-amplified antigen-specific T cells can be achieved using PCR-based T cell receptor single cell analysis methods (TCR-SCAN) (e.g., Dossinger et al., 2013). Thus, using a multimer-guided sorting strategy, T cells that selectively recognize a Hormadl peptide (e.g., SEQ ID NO: 5) can be isolated from PBMC of HLA-A2 positive patients or from T cells that have been stimulated (e.g., using a peptide or aAPC). Following infusion, antigen-specific T cells can be tracked with tetramers or multimers to assess long-term persistence in vivo.

[0101] The TCRs (or multivalent complexes thereof) of the present disclosure can alternatively or additionally be associated with (e.g., covalently or otherwise linked to) a therapeutic agent, which can be, for example, a toxic moiety for use in cell killing, or an immunostimulatory agent such as an interleukin or cytokine. The multivalent TCR complexes of the present disclosure can have enhanced binding capacity for a TCR ligand compared to non-multimeric T cell receptor heterodimers. Thus, in some embodiments, the multivalent TCR complexes can be used to track or target cells presenting a particular antigen in vitro or in vivo. The TCR or multivalent TCR complex can thus be provided in a pharmaceutically acceptable formulation for use in vivo.

[0102] The present disclosure also provides methods for delivering a therapeutic agent to a target cell, the method comprising contacting a potential target cell with a TCR or multivalent TCR complex having specificity for a TCR ligand and having associated therewith a therapeutic agent, under conditions that allow attachment of the TCR or multivalent TCR complex to the target cell.

[0103] In some implementations, soluble TCRs or multivalent TCR complexes can be used to deliver therapeutic agents to the site of cells presenting specific antigens. This can be useful, for example, for the treatment of tumors. Deliverable therapeutic agents allow them to act locally and not only on the cells to which they are bound (e.g., chemotherapeutic agents, radioactive agents, or enzymatic agents can cause localized effects near or on the tumor). Therefore, a particular strategy envisions antitumor molecules linked to T-cell receptors or multivalent TCR complexes that are specific to tumor antigens.

[0104] Many therapeutic agents can be used for this purpose, such as radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin). To reduce or limit toxicity at the desired site, the toxin can be delivered in liposomes linked to streptoacidin, allowing for slow release of the compound. This reduces damaging effects during transport in vivo and can help limit toxicity until after the TCR binds to the relevant antigen-presenting cells or cells expressing the Hormad1 antigen (e.g., cancer cells).

[0105] Other suitable therapeutic agents include: (1) small molecule cytotoxic agents, i.e., compounds capable of killing mammalian cells with a molecular weight of less than 700 Daltons. Such compounds may also contain toxic metals capable of cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also contain prodrugs, i.e., compounds that decay or transform under physiological conditions to release cytotoxic agents. Some examples of such agents include cisplatin, maytansine derivatives, rachelmycin, calcitriol, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotetamide, trimethyltroxa glucuronide, oseltamine E vincristine, and doxorubicin; (2) peptide cytotoxic agents, i.e., proteins or fragments thereof capable of killing mammalian cells. Some examples include ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNase and RNase; (3) radionuclides, which are unstable isotopes of elements that decay with the simultaneous emission of one or more of α or β particles or γ rays. Some examples include iodine-131 ( 131 I), rhenium 186( 186 Re), Indium 111 ( 111 In), Yttrium 90 ( 90 Yt), bismuth 210 and 213 ( 210 Bihe 213 Bi), Actinium 225( 225 Ac) and astatine 213 ( 213At); (4) prodrugs, such as antibody-directed enzyme prodrugs; and (5) immunostimulants, i.e., moieties that stimulate an immune response. Some examples include: cytokines, such as IL-2; chemokines, such as IL-8; platelet factor 4; melanoma growth stimulatory protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; xenogenic protein domains; allogenic protein domains; viral / bacterial protein domains, and viral / bacterial peptides.

[0106] The soluble TCRs of the present disclosure can be used to modulate T cell activation by binding to specific TCR ligands and thereby inhibiting T cell activation. Autoimmune diseases involving T cell-mediated inflammation and / or tissue damage, such as Type I diabetes, can be treated using this approach. For this use, the specific peptide epitope presented by the relevant pMHC needs to be known.

[0107] The soluble TCRs and / or multivalent TCR complexes of the present disclosure can be used to prepare a composition for treating cancer or an autoimmune disease.

[0108] Also provided are methods of treating cancer (e.g., leukemia, lung cancer, esophageal cancer, head and neck cancer, or cervical cancer, etc.) or other cancer expressing Hormadl as described herein) or an autoimmune disease, comprising administering to a patient in need thereof an effective amount of the soluble TCR and / or multivalent TCR complex of the present disclosure.

[0109] As is common in anti-cancer and autoimmune therapies, the TCRs of the present disclosure can be used in combination with other agents for treating cancer or an autoimmune disease, and one or more additional therapeutic agents or treatments can be administered to treat other related conditions found in a patient group.

[0110] C. Bispecific TCRs

[0111] In some embodiments, the TCRs of the present disclosure are comprised in a bispecific T cell receptor (TCR). Bispecific TCRs generally comprise a TCR fused, linked, or covalently bound to a scFv or antibody (e.g., McCromack et al., 2013). In some embodiments, the bispecific TCRs of the present disclosure comprise a Hormadl-directed TCR and a T cell recruiting antibody domain or scFv (e.g., a scFv against CD3 or other immunomodulatory T cell surface protein). Bispecific TCRs can cause T cells to be activated and attack tumors regardless of the T cell’s inherent specificity. Bispecific platforms that can be used with the TCRs of the present disclosure include BiTE® (Amgen, Thousand Oaks, CA). Additional examples of bispecific TCRs are ImmTACs (e.g., Oates et al., 2013).

[0112] D. Chimeric Antigen Receptors

[0113] A chimeric antigen receptor (CAR) is an engineered receptor that can be expressed by a T cell and can bind to an antigen, e.g., on a cancer cell. CARs typically comprise different domains, including an antigen binding region domain, a transmembrane domain, and an endodomain. Upon antigen recognition, the endodomain transmits activation and costimulatory signals to the T cell. Chimeric antigen receptor molecules are not naturally occurring and are distinguished by two abilities: binding to an antigen and transducing an activation signal through an immunoreceptor activation motif (ITAM)-containing motif in its cytoplasmic endodomain. CAR T cells are T cells that have been genetically modified to express a CAR.

[0114] A soluble TCR construct can be fused to a CAR signaling tail (i.e., a transmembrane domain and an endodomain) to direct T cell recognition of an antigen, e.g., as described in Walseng et al. (2017). Such a CAR construct is referred to as a “TCR-CAR.” The CAR can thus comprise a TCR binding region (e.g., as shown in FIGS. 6A-6B the disclosure covalently linked to a transmembrane domain and an endodomain, or expressed as a fusion protein with a transmembrane domain and an endodomain. The endodomain can comprise, e.g., CD3 zeta, CD28 intracellular signaling domain, 4-1BB (CD137), (CD3 zeta and CD28), CD27, OX-40 (CD134), DAP10, or 4-1BB.

[0115] II. Adoptive Cell Transfer Therapy

[0116] Provided herein are methods for treating or delaying progression of a cancer in an individual comprising administering to the individual an effective amount of an antigen-specific immune cell or stem cell (e.g., an autologous or allogeneic T cell (e.g., a regulatory T cell, CD4+ T cell, CD8+ T cell, alpha-beta T cell, or gamma-delta T cell), NK cell, invariant NK cell, NKT cell, mesenchymal stem cell (MSC), or induced pluripotent stem (iPS) cell) therapy, e.g., a Hormad1 -specific cell therapy. Also provided herein are adoptive T cell therapies with T cells transduced with a genetically engineered TCR (e.g., expressing a TCR comprising one or more of SEQ ID NOs: 1-4, e.g., SEQ ID NO: 2 and SEQ ID NO: 4). In some embodiments, the adoptive cell transfer therapy is provided to a subject (e.g., a human patient) in combination with a second therapy, e.g., a chemotherapy, a radiation therapy, a surgery, or a second immunotherapy.

[0117] The peptides provided herein (e.g., SEQ ID NO: 5) can also be used to generate antigen-specific cytotoxic T cell (CTL) cell lines or clones that can be used for adoptive immunotherapy. The peptides or corresponding polynucleotides encoding the peptides can be loaded onto dendritic cells, lymphoblastoid cell lines (LCLs), PBMCs, or artificial antigen presenting cells (aAPCs) and then co-cultured with T cells for several rounds of stimulation to generate antigen-specific CTL cell lines or clones (e.g., Neal et al., 2017). A variety of antigen presenting cells (APCs) can be used to expand T cells ex vivo, and a variety of strategies for antigen loading of dendritic cells can be used to enhance anti-tumor responses (see, e.g., Strome et al., 2002). The resulting autologous CTL cell lines or clones can be used for adoptive cell transfer immunotherapy for the treatment of cancer patients.

[0118] Some embodiments of the present disclosure include methods of obtaining autologous T cells from a subject, methods of making TCR-engineered immune cells or stem cells, and methods of administering TCR-engineered cells to a subject as an immunotherapy targeting cancer cells. In particular, the TCR-engineered immune cells or stem cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, or gamma-delta T cells), NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), or induced pluripotent stem (iPS) cells) are antigen-specific cells (e.g., Hormad1-specific cells). Over the past two decades, several basic approaches for the derivation, activation, and expansion of functional anti-tumor effector cells have been described. 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 capture of target cell membranes; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells that are genetically reprogrammed or “redirected” to express tumor-reactive TCR or chimeric TCR molecules, known as “T-bodies,” that exhibit antibody-like tumor recognition capabilities (e.g., Eshhar et al., 1995). These approaches have generated a number of protocols for T cell preparation and immunization that can be used in the methods described herein.

[0119] A. T cell preparation and administration

[0120] In some embodiments, the engineered T cells are autologous (i.e., isolated from the patient to be treated). In some embodiments, the engineered T cells are allogeneic. In some embodiments, the allogeneic T cells comprise T cells pooled from multiple donors.

[0121] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or a lymphoid organ. The T cells are most preferably human cells. In some embodiments, T cells obtained from umbilical cord blood can have improved anti-tumor properties compared to T cells obtained from adult donors (e.g., Hiwarkar et al., 2015). The cells are typically primary cells, e.g., those isolated directly from a subject and / or isolated from a subject and frozen. In some embodiments, the cells comprise one or more subpopulations of T cells or another cell type, e.g., T cells from whole blood, CD4+ cells, CD8+ cells, and subpopulations thereof, e.g., those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization and / or persistence capacity, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. The cells can be allogeneic and / or autologous with respect to the subject to be treated. In some aspects, e.g., for off-the-shelf technologies, the cells are pluripotent and / or multipotent, e.g., stem cells, e.g., induced pluripotent stem (iPS) cells; e.g., the stem cells or iPS cells can be differentiated into multiple T cell populations. In some embodiments, the methods comprise isolating cells from a subject, preparing, treating, culturing, and / or engineering them as described herein, and reintroducing them into the same patient (if they are autologous) or a different patient (if they are allogeneic) before or after cryopreservation.

[0122] T cells (e.g., CD4 + and / or CD8 + T cells) exist in subtypes and subpopulations of naive T (T N ) cells, effector T cells (T EFF ), memory T cells (T MEM ) and subtypes thereof (e.g., stem cell memory T (TSC M ) cells, central memory T (TC M ) cells, effector memory T (T EM ) cells, or terminally differentiated effector memory T cells (T EMRAT cells derived from tumor-infiltrating lymphocytes (TILs), including immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and 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), α / β T cells, and δ / γ T cells.

[0123] In some implementations, T cell subsets can be generated by isolating, enriching, or depleting cells that are positive or negative for specific markers, such as cell surface markers. In some cases, such markers are those that are absent or expressed at relatively low levels in certain T cell populations (e.g., non-memory cells) but present or expressed at relatively high levels in certain other T cell populations (e.g., memory cells).

[0124] In some implementations, T cells are isolated from PBMC samples by negative selection of markers expressed on non-T cells (e.g., B cells, monocytes, or other leukocytes). In some aspects, CD4... + or CD8 + Select the step for separating CD4 + Support and CD8 + Cytotoxic T cells. CD4+ is a marker expressed or expressed at relatively high levels on one or more subsets of naive, memory, and / or effector T cells through positive or negative selection. + and CD8 + The population can be further sorted into subpopulations. Several methods can be used for marker-based cell isolation, including magnetically activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS).

[0125] In some implementations, such as by positive or negative selection based on surface antigens associated with the corresponding subgroups, CD8 + T cells further enrich or deplete naïve, central memory, effector memory, and / or central memory stem cells. In some implementations, central memory T(T) cells are used. CM Enrichment of cells to improve potency, for example, to improve long-term survival, expansion and / or implantation after administration (e.g., see Terakura et al., 2012; Wang et al., 2012).

[0126] In some embodiments, the T cells are autologous T cells. In this method, a biological sample (e.g., a blood sample or a bone marrow sample) is obtained from the patient. In some embodiments, the cell suspension or culture is prepared from a biological sample obtained from the patient (e.g., obtained from a tumor). The single cell suspension can be obtained in any suitable manner, e.g., mechanically (e.g., using, e.g., a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif. to dissociate the tumor) or enzymatically (e.g., using collagenase or DNase). The single cell suspension of the tumor enzyme digest is cultured in interleukin 2 (IL-2). The cells are cultured until confluent (e.g., about 2 x 10 TM lymphocytes), e.g., about 5 to about 21 days, preferably about 10 to about 14 days. For example, the cells can be cultured from 5 days, 5 to 6 days, or 5 to 21 days, or 10 to 14 days. 6

[0127] In some embodiments, naked DNA or a suitable vector encoding a TCR or CAR of the disclosure can be introduced into T cells of a subject (e.g., T cells obtained from a human patient having a cancer or other disease). Methods of using naked DNA to stably transfect T cells by electroporation are known in the art. See, e.g., U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor of the invention contained in a plasmid expression vector in the appropriate expression orientation (e.g., Zhang et al., 2018). In some embodiments, the use of naked DNA can reduce the time required to generate T cells expressing a TCR generated by the methods of the invention. Transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009 can be used. Electroporation of RNA encoding full-length TCR a and β (or γ and δ) chains can be used as an alternative to overcome the long-term problem of self-reactivity caused by retroviral transduction and endogenous TCR chain pairing. In some embodiments, non-viral RNA transfection can be used to transiently modify T cells, e.g., as described in Riet et al. (Methods Mol Biol. 2013; 969: 187-201).

[0128] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce a TCR or chimeric construct into a T cell. In general, a vector encoding a TCR or CAR used to transfect T cells from a subject should generally be non-replicative in the T cells of the subject. A large number of vectors are known based on viruses, where the number of viral copies maintained in the cell is low enough to maintain the viability of the cell. Some exemplary vectors include the pFB-neo vector ​and vectors based on HIV, SV40, EBV, HSV, or BPV.

[0129] In some embodiments, TCR nucleotide sequences (e.g., DNA or RNA sequences) encoding the alpha and beta chains of the present disclosure (e.g., see FIGS. 6A-6B ; SEQ ID NOs: 1-4) can be cloned into a retroviral, lentiviral, or other expression vector, such as MSCV (murine stem cell virus) or a plasmid (e.g., adeno-associated virus-derived plasmid). T cells can be genetically altered to express the TCR. PBMCs are a source of both antigen presenting cells and T cells. T cells expressing the TCR can be used in adoptive cell transfer therapy for cancer patients.

[0130] Once it is determined that the transfected or transduced T cells are capable of expressing the TCR or CAR as a surface membrane protein and at a desired level, it can be determined whether the TCR or chimeric receptor is functional in the host cell to provide the desired signal induction. Subsequently, the transduced T cells can be reintroduced into or administered to a subject to activate, implement, and / or elicit an anti-tumor response in the subject. To facilitate administration, the transduced T cells can be made into a pharmaceutical composition with an appropriate pharmaceutically acceptable carrier or diluent or into an implant suitable for in vivo administration. Methods of making such compositions or implants have been described in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 22ndedition, Pharmaceutical Press, 2012). Where appropriate, the transduced T cells expressing the TCR or CAR can be formulated into preparations in solid or liquid form, e.g., capsules, solutions, injectables, in a common way for their respective routes of administration. Methods known in the art can be utilized to retard or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed release of the composition. In general, it is preferred to employ a pharmaceutically acceptable form that does not significantly adversely affect the cells expressing the TCR or chimeric receptor. In some embodiments, the transduced T cells can be made into a pharmaceutical composition comprising a balanced salt solution, such as Hanks' balanced salt solution or physiological saline. nd edition, Pharmaceutical Press, 2012). Where appropriate, the transduced T cells expressing the TCR or CAR can be formulated into preparations in solid or liquid form, e.g., capsules, solutions, injectables, in a common way for their respective routes of administration. Methods known in the art can be utilized to retard or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed release of the composition. In general, it is preferred to employ a pharmaceutically acceptable form that does not significantly adversely affect the cells expressing the TCR or chimeric receptor. In some embodiments, the transduced T cells can be made into a pharmaceutical composition comprising a balanced salt solution, such as Hanks' balanced salt solution or physiological saline.

[0131] T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50, 60, 70, 80, 90, or 100-fold or more) over a period of about 10 to about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900-fold or higher) over a period of about 10 to about 14 days. In some embodiments, allogeneic T cells can be pooled from several donors.

[0132] Expansion can be accomplished by a variety of methods known in the art. For example, T cells can be rapidly expanded using non-specific TCR stimulation in the presence of feeder layer lymphocytes and interleukin 2 (IL-2) or interleukin 15 (IL-15), with IL-2 being preferred. Non-specific TCR stimulation can comprise about 30 ng / ml of OKT3 (mouse monoclonal anti-CD3 antibody, available from Ortho-Clinical Diagnostics, Raritan, N.J.). Alternatively, T cells can be rapidly expanded by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more antigens of a cancer, including antigenic portions thereof, e.g., epitopes, or cells, which can optionally be expressed from a vector, e.g., a human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T cell growth factor, e.g., 300 IU / ml IL-2 or IL-15, with IL-2 being preferred. In vitro induced T cells are rapidly expanded by restimulation with the same cancer antigen pulsed onto an antigen presenting cell expressing HLA-A2. Alternatively, T cells can be restimulated, e.g., with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes, and IL-2. Raritan, N.J. obtained) Alternatively, T cells can be rapidly expanded by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more antigens of a cancer, including antigenic portions thereof, e.g., epitopes, or cells, which can optionally be expressed from a vector, e.g., a human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T cell growth factor, e.g., 300 IU / ml IL-2 or IL-15, with IL-2 being preferred. In vitro induced T cells are rapidly expanded by restimulation with the same cancer antigen pulsed onto an antigen presenting cell expressing HLA-A2. Alternatively, T cells can be restimulated, e.g., with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes, and IL-2.

[0133] Autologous T cells can be modified to express a T cell growth factor that promotes growth and activation of the autologous T cells. Suitable T cell growth factors include, e.g., interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable methods of modification are known in the art, including, e.g., Sambrook et al., 2001; and Ausubel et al., 1994. In some embodiments, the modified autologous T cells express the T cell growth factor at a high level. T cell growth factor coding sequences, e.g., that of IL-12, are readily available in the art, as are promoters that can be used to promote high level expression.

[0134] In certain embodiments, a T cell growth factor that promotes growth and activation of autologous or allogeneic T cells is administered to the subject concurrently with or after the autologous T cells. The T cell growth factor can be any suitable growth factor that promotes growth and activation of autologous T cells. Some examples of suitable T cell growth factors include interleukin (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 is a preferred T cell growth factor.

[0135] The T cells can be administered intravenously, intramuscularly, subcutaneously, transdermally, intraperitoneally, intrathecally, parenterally, intrathecally, intraluminally, intraventricularly, intra-arterially, through cerebrospinal fluid, or through any implantable or semi-implantable, permanent or degradable device. Suitable dosages for T cell therapy can be determined based on the type of disease to be treated, the severity and course of the disease, the individual's clinical history and response to treatment, and the judgment of the attending physician.

[0136] Injection into a tumor or into the tumor vasculature is specifically contemplated for discrete, accessible solid tumors. Local, regional, or systemic administration can also be appropriate. For tumors >4 cm, a volume of about 4 to 10 ml (particularly 10 ml) can be administered, while for tumors <4 cm, a volume of about 1 to 3 ml (e.g., 3 ml) can be used. Multiple injections delivered as a single dose can comprise a volume of about 0.1 to about 0.5 ml.

[0137] B. Antigen Presenting Cells

[0138] Antigen presenting cells (APCs) are a heterogeneous group of immune cells that mediate cellular immune responses by processing and presenting antigens for recognition by certain lymphocytes, such as T cells. APCs include dendritic cells, macrophages, Langerhans cells, and B cells. APCs can process protein antigens, breaking them down into peptides, and present them on the cell surface along with major histocompatibility complex (MHC) molecules, where they can interact with appropriate T cell receptors. APCs are distinguished by their expression of specific MHC molecules. The MHC is a large genetic complex with multiple loci. The MHC loci encode two major types of MHC membrane molecules, called class I and class II MHC. T helper lymphocytes typically recognize antigens associated with MHC class II molecules, while T cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. In humans, the MHC is known as the HLA complex, and in mice it is known as the H-2 complex.

[0139] In some embodiments, the peptide (e.g., SEQ ID NO: 5) is recognized by HLA-A2 and can be used to expand antigen-specific T cells in vitro. The peptide or nucleic acid encoding the peptide can be used to stimulate antigen presenting cells (APCs) to trigger the initiation of an immune response. In some embodiments, the peptide or the corresponding polynucleotide encoding the peptide can be loaded onto dendritic cells, lymphoblastoid cell lines (LCLs), PBMCs, or artificial antigen presenting cells (aAPCs) and subsequently co-cultured with T cells for several rounds of stimulation to generate an antigen-specific CTL cell line or clone. Thus, an expanded population of T cells that selectively recognize the Hormadl -derived peptide / HLA-A2 complex can be adoptively transferred to a patient to treat cancer or induce tumor regression.

[0140] In some cases, artificial antigen presenting cells (aAPCs) can be used to make TCR or CAR-based therapeutic compositions and cell therapy products. For general guidance regarding the preparation and use of antigen presenting systems, see, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662; U.S. Patent Application Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. WO 2007 / 103009.

[0141] aAPCs can be used to expand T cells expressing TCRs or CARs. The signals delivered to T cells by antigen-presenting cells during encounters with tumor antigens influence T cell programming and their subsequent therapeutic potency. This has stimulated efforts to develop artificial antigen-presenting cells that allow optimal control over the signals provided to T cells (Turtle et al., 2010). In addition to the antibody or antigen of interest, the aAPC system can comprise at least one exogenous accessory molecule. Any suitable number and combination of accessory molecules can be used. The accessory molecules can be costimulatory molecules or adhesion molecules. Exemplary costimulatory molecules include CD70 and B7.1 (also known as B7 or CD80), which can bind to CD28 and / or CTLA-4 molecules on the surface of T cells, thereby promoting, for example, T cell expansion, Thl differentiation, short-term T cell survival, and cytokine secretion, such as interleukin (IL)-2 (see Kim et al., 2004). Adhesion molecules can include: carbohydrate-binding glycoproteins, such as selectins; transmembrane binding glycoproteins, such as integrins; calcium-dependent proteins, such as cadherins; and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), which promote, for example, cell-to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for selecting, cloning, making, and expressing exemplary accessory molecules, including costimulatory molecules and adhesion molecules, are exemplified in, e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001.

[0142] C. Nucleic Acids

[0143] In one aspect, the present disclosure provides nucleic acids encoding the isolated TCRs (e.g., sTCRs), CARs, or peptides disclosed herein. For example, the nucleic acids can encode a polypeptide comprising a TCR variable region having about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a TCR variable region disclosed herein (e.g., SEQ ID NOs: 1-4), or a TCR variable region having 1, 2, 3, or 4 point mutations (e.g., substitution mutations) compared to any one of SEQ ID NOs: 1-4. The term “nucleic acid” is intended to include DNA and RNA and can be double-stranded or single-stranded.

[0144] Accordingly, nucleic acids encoding a TCR (e.g., sTCR), CAR, or peptide can be operably linked to a promoter and / or contained in an expression vector. Methods well known in the molecular biology art can be used to produce a TCR, CAR, or peptide in a suitable expression system. Nucleic acids encoding a tumor antigen-specific peptide disclosed herein can be incorporated into any expression vector that ensures good expression of the peptide in the desired environment (e.g., in a human immune cell). Possible vectors that can be used 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 the host cell.

[0145] A recombinant expression vector that is "suitable for transformation of a host cell" means that the expression vector comprises a nucleic acid molecule of the disclosure and regulatory sequences selected based on the host cell to be used for expression operatively linked to the nucleic acid molecule. The terms "operatively linked" or "operably linked" are used interchangeably and are intended to mean that the nucleic acid is linked to the regulatory sequences in a manner that allows for expression of the nucleic acid under the control of the regulatory sequences.

[0146] Accordingly, the present disclosure provides a recombinant expression vector comprising a nucleic acid encoding a TCR, CAR, or soluble peptide that selectively binds to Hormad1, and the necessary regulatory sequences for transcription and translation of the inserted protein sequence. Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, or viral genes (see, e.g., the regulatory sequences described in Goeddel, 1990).

[0147] The selection of appropriate regulatory sequences typically depends on the host cell selected, and can be readily accomplished by one of ordinary skill in the art. Some examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence; a ribosomal binding sequence including a translation initiation signal. Additionally, other sequences, such as a replication origin, additional DNA restriction sites, enhancers, and sequences which

[0148] The recombinant expression vector can also comprise a selectable marker gene that facilitates selection of host cells transformed or transfected with a TCR, CAR, or soluble peptide that selectively binds to Hormadl disclosed herein. Some examples of selectable marker genes are genes encoding proteins that confer resistance to certain drugs, such as G418 and hygromycin, beta-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in concentration of the selectable marker protein (e.g., beta-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase). If the selectable marker gene encodes a protein that confers resistance to an antibiotic, such as neomycin, transformed cells can be selected with G418 (Geneticin); thus, cells that have incorporated the selectable marker gene will survive while other cells die. This allows visualization and determination of the expression of the recombinant expression vector, and also allows determination of the effect of mutations on expression and phenotype.

[0149] The recombinant expression vector can also comprise a selectable marker gene that facilitates selection of host cells transformed or transfected with a TCR, CAR, or soluble peptide that selectively binds to Hormadl disclosed herein. Some examples of selectable marker genes are genes encoding proteins that confer resistance to certain drugs, such as G418 and hygromycin, beta-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in concentration of the selectable marker protein (e.g., beta-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase). If the selectable marker gene encodes a protein that confers resistance to an antibiotic, such as neomycin, transformed cells can be selected with G418 (Geneticin); thus, cells that have incorporated the selectable marker gene will survive while other cells die. This allows visualization and determination of the expression of the recombinant expression vector, and also allows determination of the effect of mutations on expression and phenotype.

[0150] The nucleic acid molecules of the present disclosure can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polynucleotides are known, including solid phase synthesis, which is analogous to peptide synthesis, and has been automated in commercially available DNA synthesizers (see, e.g., U.S. Pat. Nos. 4,598,049; 4,458,066; 4,401,796; and 4,373,071).

[0151] III. Peptide Vaccines

[0152] In some aspects, methods for treating cancer (e.g., breast cancer, lung cancer, etc.) are provided that include immunizing a subject with a purified tumor antigen or immunodominant tumor antigen specific peptide, such as a Hormadl peptide (SEQ ID NO: 5). The Hormadl peptide can be administered to a mammalian subject, such as a human patient, by a variety of routes (e.g., intramuscular, intravenous, subcutaneous, etc.). In some embodiments, the peptide can be injected in solution (e.g., saline solution) as a vaccine or to elicit an immune response against the peptide. For example, to improve solubility of the peptide and / or to improve the immune response in the subject, an adjuvant can be included in the formulation or solution (e.g., as described in Massarelli et al., 2019). In some embodiments, peptide-pulsed mature dendritic cells can be administered to the subject. Methods that can be used to elicit an immune response or anti-cancer response against the peptide in a subject include, for example, those described in Wen et al. (2019) and Massarelli et al. (2019). In some embodiments, the Hormadl peptide (SEQ ID NO: 5) is combined with or presented by autologous dendritic cells that can be reinfused into the subject or human patient.

[0153] IV. Anti-cancer therapy

[0154] Some embodiments of the present disclosure relate to administering an additional anti-cancer therapeutic. In some embodiments, the additional anti-cancer therapy is one described herein. Some examples of additional anti-cancer therapeutics are provided below.

[0155] A. Immune stimulants

[0156] In some embodiments, the method further comprises administering an additional agent. In some embodiments, the additional agent is an immunostimulant. As used herein, the term "immunostimulant" refers to a compound that can stimulate an immune response in a subject, and can include an adjuvant. In some embodiments, the immunostimulant is a substance that does not constitute a specific antigen but can enhance the intensity and persistence of the immune response to an antigen. Such immunostimulants can include, but are not limited to: stimulators of pattern recognition receptors (e.g., Toll-like receptors, RIG-1, and NOD-like receptors (NLR)); mineral salts, such as alum, with monphosphoryl lipid (MPL) A of Enterobacteria (e.g., E. coli), Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri, or, in particular, alum combined with MPL.RTM. (AS04), MPL A of the aforementioned bacteria alone; saponins, such as QS-21, Quil-A, ISCOM, ISCOMATRIX; emulsions, such as MF59, Montanide, ISA51 and ISA 720, AS02 (QS21 + squalene + MPL.); liposomes and liposomal formulations, such as AS01; synthetic or specially prepared microparticles and microcarriers, such as outer membrane vesicles (OMVs) of bacterial origin from N. gonorrheae, Chlamydia trachomatis, etc.; or chitosan particles; depot-forming agents, such as pluronics block copolymers; specifically modified or prepared peptides, such as muramyl dipeptide; aminoalkyl amino glucoside 4-phosphates, such as RC529; or proteins, such as bacterial toxoids or toxin fragments.

[0157] In some embodiments, the additional agent comprises an agonist of a pattern recognition receptor (PRR), including but not limited to a Toll-like receptor (TLR), in particular TLR2, 3, 4, 5, 7, 8, 9, and / or combinations thereof. In some embodiments, the additional agent comprises an agonist of Toll-like receptor 3, an agonist of Toll-like receptors 7 and 8, or an agonist of Toll-like receptor 9; preferably, the recited immunostimulatory agent comprises an imidazoquinoline; e.g., R848; an adenine derivative, e.g., those disclosed in U.S. Patent No. 6,329,381, U.S. Published Patent Application 2010 / 0075995, or WO 2010 / 018132; an immunostimulatory DNA; or an immunostimulatory RNA.In some embodiments, the additional agent can also comprise an immunostimulatory RNA molecule, such as, but not limited to, dsRNA, poly I:C or poly I:poly C12U (available as Ampligen.RTM, both poly I:C and poly I:poly C12U are known as TLR3 stimulators) and / or those disclosed in F. Heil et al., "Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer et al., "Immune modulation by chemically modified ribonucleosides and oligoribonucleotides" WO2008033432A2; A. Forsbach et al., "Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targeting the Toll-like receptor 8 pathway" WO 2007062107 A2; E. Uhlmann et al., "Modified oligoribonucleotide analogs with enhanced immunostimulatory activity" U.S. Patent Application Publication US 2006241076; G. Lipford et al., "Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections" WO 2005097993A2; G. Lipford et al., "Immunostimulatory G, U-containing oligoribonucleotides, compositions, and screening methods" WO 2003086280A2. In some embodiments, the additional agent can be a TLR-4 agonist, such as, for example, bacterial lipopolysaccharide (LPS), VSV-G and / or HMGB-1.In some embodiments, the additional agent can comprise a TLR-5 agonist, such as flagellin, or a portion or derivative thereof, including but not limited to those disclosed in U.S. Patent Nos. 6,130,082, 6,585,980, and 7,192,725.

[0158] In some embodiments, the additional agent can be a pro-inflammatory stimulus released from necrotic cells (e.g., urate crystals). In some embodiments, the additional agent can be an activating component of the complement cascade (e.g., CD21, CD35, etc.). In some embodiments, the additional agent can be an activating component of immune complexes. Additional agents also include complement receptor agonists, such as molecules that bind to CD21 or CD35. In some embodiments, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarriers. In some embodiments, the immune stimulant is a cytokine, which is a small protein or biological factor (in the range of 5 kD to 20 kD) that is released by cells and has specific effects on cell-cell interactions, communication, and the behavior of other cells. In some embodiments, the cytokine receptor agonist is a small molecule, an antibody, a fusion protein, or an aptamer.

[0159] B. Immunotherapy

[0160] In some embodiments, the additional therapy comprises cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be classified as active, passive, or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, called tumor-associated antigens (TAAs); they are usually proteins or other macromolecules (such as carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapy augments existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Immunotherapy is known in the art, and some are described below.

[0161] 1. Inhibition of costimulatory molecules

[0162] In some embodiments, the immunotherapy comprises an inhibitor of a costimulatory molecule. In some embodiments, the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. The inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0163] 2. Dendritic cell therapy

[0164] Dendritic cell therapy elicits an anti-tumor response by having dendritic cells present tumor antigens to lymphocytes, which activates them, eliciting them to kill other cells presenting the antigen. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer therapy, they help target cancer antigens. One example of dendritic cell-based cell cancer therapy is sipuleucel-T.

[0165] One way to induce dendritic cells to present tumor antigens is through vaccination with autologous tumor lysate or short peptides (small portions of proteins corresponding to protein antigens on cancer cells). These peptides are often given in combination with an adjuvant (a highly immunogenic substance) to increase the immune and anti-tumor response. Other adjuvants include proteins or other chemicals that attract or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0166] Dendritic cells can also be activated in vivo by having tumor cells express GM-CSF. This can be achieved by genetically engineering the tumor cells to produce GM-CSF, or by infecting the tumor cells with an oncolytic virus that expresses GM-CSF.

[0167] Another strategy is to remove dendritic cells from a patient's blood and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which can be a single tumor-specific peptide / protein or a tumor cell lysate (a solution that breaks down tumor cells). These cells, with optional adjuvants, are infused and elicit an immune response.

[0168] Dendritic cell therapy includes the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies and can induce dendritic cell maturation and provide immunity to tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8, or CD40 have been used as antibody targets.

[0169] 3. CAR-T cell therapy

[0170] Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T-cell receptors, or artificial T-cell receptors) are engineered receptors that combine new specificity with immune cells to target cancer cells. Generally, these receptors graft the specificity of monoclonal antibodies onto T cells. The receptor is called chimeric because it is fused from parts from different sources. CAR-T cell therapy refers to a treatment for cancer using such transformed cells.

[0171] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen binding and T cell activation functions. The general premise of CAR-T cells is that T cells are artificially generated to target markers present on cancer cells. Scientists can take T cells from a person, genetically alter them, and put them back into the patient to make them attack cancer cells. Once the T cells are engineered into CAR-T cells, they can act as a “living drug.” CAR-T cells make a connection between an extracellular ligand recognition domain and an intracellular signaling molecule, which in turn activates the T cell. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of CAR-T cell therapy safety is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule targeted.

[0172] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtageneciloleucel (Yescarta). In some embodiments, the CAR-T therapy targets CD19.

[0173] 4. Cytokine therapy

[0174] Cytokines are proteins produced by many types of cells that are present within a tumor. They can modulate the immune response. Tumors often employ them to grow and reduce the immune response. These immune modulating effects make them useful as drugs to prime the immune response. Two commonly used cytokines are interferons and interleukins.

[0175] Interferons are produced by the immune system. They are often involved in antiviral responses, but also have uses for cancer. They are divided into three groups: Type I (IFNa and IFNp), Type II (IFNy), and Type III (IFNp).

[0176] Interleukins have a range of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0177] 5. Adoptive T cell therapy

[0178] Adoptive T cell therapy is a form of passive immunization by infusion of T cells (adoptive cell transfer). They are found in blood and tissues and are normally activated when they find a foreign pathogen. Specifically, when the surface receptors of T cells encounter a cell displaying part of a foreign protein on its surface antigen, they become activated. These can be infected cells, or antigen presenting cells (APCs). They are found in normal tissues and in tumor tissues, where they are called tumor infiltrating lymphocytes (TILs). They are activated in the presence of APCs, such as dendritic cells presenting tumor antigens. Although these cells can attack tumors, the environment within tumors is highly immunosuppressive, which prevents immune-mediated tumor death.

[0179] Various ways of generating and obtaining T cells that target tumors have been developed. T cells specific for tumor antigens can be removed from tumor samples (TILs), or filtered from blood. Subsequent activation and culture is performed ex vivo, and the result is re-infused. Activation can be performed by gene therapy or by exposing T cells to tumor antigens.

[0180] 6. Checkpoint inhibitors and combination therapies

[0181] In some embodiments, the additional therapy comprises an immune checkpoint inhibitor. Certain embodiments are further described below.

[0182] PD-1 can play a role in the tumor microenvironment where T cells encounter infections or tumors. Activated T cells upregulate PD-1 and continue to express it in peripheral tissues. Cytokines, such as IFN-γ, induce expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery during immune responses and prevent excessive damage to tissues. Inhibitors of the present disclosure can block one or more functions of PD-1 and / or PDL1 activity.

[0183] Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0184] In some embodiments, a PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In one specific aspect, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partner. In one specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partner. In one specific aspect, the PDL2 binding partner is PD-1. The inhibitor may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors used in the methods and compositions provided herein are known in the art, for example, as described in U.S. Patent Applications Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0185] In some embodiments, the PD-1 inhibitor 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 nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesive (e.g., an immunoadhesive comprising the extracellular portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence) or the PD-1 binding portion). In some embodiments, the PDL1 inhibitor comprises AMP-224. Nivolumab (also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and...) ) is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab (also known as MK-3475, Merck 3475, lambrolizumab) SCH-900475 is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab (also known as CT-011, hBAT, or hBAT-1) is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224 (also known as B7-DCIg) is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Other PD-1 inhibitors include MEDI0680, also known as AMP-514 and REGN2810.

[0186] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor, e.g., Durvalumab, also known as MEDI4736; atezolizumab, also known as MPDL3280A; avelumab, also known as MSB00010118C, MDX-1105, BMS-936559; or a combination thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor, e.g., rHIgM12B7.

[0187] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2, and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any range derivable therein) variable region amino acid sequence identity to the above-mentioned antibodies.

[0188] 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 bound to B7-1 (CD80) or B7-2 (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 an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and can be important to their function. T cell activation through the T cell receptor and CD28 leads to an increased expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitors of the present disclosure can block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some embodiments, the inhibitor blocks CTLA-4 interaction with B7-1. In some embodiments, the inhibitor blocks CTLA-4 interaction with B7-2.

[0189] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human, humanized, or chimeric antibody), antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide thereof.

[0190] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the present application can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, anti-CTLA-4 antibodies disclosed in US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), US Patent No. 6,207,156; Hurwitz et al., 1998, 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 No. WO 2001 / 014424, WO 2000 / 037504, and US Patent No. 8,017,114; all incorporated herein by reference.

[0191] An additional anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the present disclosure is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). ) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424).

[0192] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any range derivable therein) variable region amino acid sequence identity with the above-mentioned antibodies.

[0193] C. Oncolytic Viruses

[0194] In some embodiments, the additional therapy comprises an oncolytic virus. Oncolytic viruses are viruses that preferentially infect and kill cancer cells. When infected cancer cells are destroyed by oncolysis, they release new infectious viral particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought to cause not only direct destruction of tumor cells, but also to stimulate the host's anti-tumor immune response for long-term immunotherapy.

[0195] D. Polysaccharides

[0196] In some embodiments, the additional therapy comprises a polysaccharide. Certain compounds found in mushrooms, primarily polysaccharides, can upregulate the immune system and can have anti-cancer properties. For example, beta-glucans (e.g., lentinan) have been shown in laboratory studies to stimulate macrophages, NK cells, T cells, and immune system cytokines, and have been studied in clinical trials as an immunologic adjuvant.

[0197] E. Neoantigens

[0198] In some embodiments, the additional therapy comprises neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for T cell immunotherapy. The presence of CD8+ T cells in cancer lesions is higher in tumors with high mutational burden, as determined using RNA sequencing data. Transcriptional levels associated with cytolytic activity of natural killer cells and T cells are positively correlated with mutational burden in many human tumors.

[0199] F. Chemotherapy

[0200] In some embodiments, the additional therapy comprises chemotherapy. Suitable classes of chemotherapeutic agents include: (a) alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin), and triazines (e.g., dacarbazine); (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine), and purine analogs and related agents (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); (c) natural products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-α); and (d) other agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0201] Cisplatin has been widely used to treat cancer, such as metastatic testicular or ovarian cancer, advanced bladder cancer, head and neck cancer, cervical cancer, lung cancer, or other tumors. Cisplatin is not absorbed orally and thus must be delivered by other routes, such as intravenous, subcutaneous, intratumoral, or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, and in certain embodiments, an effective dose contemplated for use in clinical applications includes: about 15 mg / m2to about 20 mg / m2for 5 days every three weeks, for a total of three courses. In some embodiments, the amount of cisplatin delivered to a cell and / or subject in combination with a construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount that would be delivered if cisplatin were used alone.

[0202] Other suitable chemotherapeutic agents include anti-microtubule agents, such as paclitaxel ("Taxol") and doxorubicin hydrochloride ("Doxil"). The determination that the combination of the Egr-1 promoter / TNFa construct delivered by an adenoviral vector and doxorubicin is effective in overcoming resistance to chemotherapeutics and / or TNF-a indicates that the combination therapy of the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-a.

[0203] Doxorubicin is poorly absorbed and is preferably administered intravenously. In certain embodiments, suitable intravenous doses for an adult human include: about 60 mg / m2to about 75 mg / m2at intervals of about 21 days; or about 25 mg / m2to about 30 mg / m2repeated every other week for 2 or 3 consecutive days; or about 20 mg / m2once a week. In older patients, the lowest dose should be used when there is prior myelosuppression from prior chemotherapy or neoplastic marrow invasion or when the drug is combined with other myelosuppressive drugs.

[0204] Nitrogen mustards are another suitable chemotherapeutic agent that can be used in the methods of the disclosure. Nitrogen mustards can include, but are not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (Cytoxan®) Available from Mead Johnson, and Available from Adria) is another suitable chemotherapeutic agent. Suitable oral doses for an adult human include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous doses include, for example, initial divided doses of about 40 mg / kg to about 50 mg / kg over a period of about 2 days to about 5 days, or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days, or about 3 mg / kg to about 5 mg / kg twice a week, or about 1.5 mg / kg / day to about 3 mg / kg / day. Intravenous routes are preferred due to adverse gastrointestinal effects. The drug is also sometimes administered intramuscularly by infiltration or into body cavities.

[0205] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU), and floxuridine (floxuridine; FudR). 5-FU can be administered to a subject at any dose between about 7.5 to about 1000 mg / m2. Further, 5-FU dosing regimens can be over a variety of time periods, such as up to six weeks, or as determined by one of ordinary skill in the art to which the disclosure pertains.

[0206] Another suitable chemotherapeutic agent, gemcitabine diphosphate Eli Lilly & Co., "Gemzar") is recommended for the treatment of advanced and metastatic pancreatic cancer, and thus will also be useful in the present disclosure for these cancers.

[0207] The amount of chemotherapeutic agent delivered to the patient can be variable. In one suitable embodiment, when the chemotherapy is administered with the construct, the chemotherapeutic agent can be administered in an amount effective to cause a halt or regression of the cancer in the host. In other embodiments, the chemotherapeutic agent can be administered in any amount between 2 to 10,000 fold less than the chemotherapeutically effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent can be administered in an amount that is about 20 fold less, about 500 fold less, or even about 5000 fold less than the chemotherapeutically effective dose of the chemotherapeutic agent. The chemotherapeutic agents of the present disclosure can be tested in vivo in combination with the construct for the desired therapeutic activity, as well as for determining effective doses. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chickens, cows, monkeys, rabbits, and the like. In vitro testing can also be used to determine suitable combinations and doses, as described in the Examples.

[0208] G. Radiation Therapy

[0209] In some embodiments, the additional or prior treatment comprises radiation, e.g., ionizing radiation. As used herein, "ionizing radiation" is meant to include radiation that includes particles or photons that have sufficient energy or can produce sufficient energy by nuclear interaction to produce ionization (the gain or loss of electrons). One exemplary and preferred ionizing radiation is x-radiation. Means for delivering x-radiation to target tissues or cells are well known in the art.

[0210] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 40 Gy (or any range derivable therein). In some embodiments, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any range derivable therein). When more than one dose is administered, the doses can be separated by about 1, 4, 8, 12, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks, or any range derivable therein.

[0211] In some embodiments, the amount of IR can be expressed as a total dose of IR, which is then administered in fractional doses. For example, in some embodiments, the total dose is 50 Gy, administered in 10 fractional doses of 5 Gy each. In some embodiments, the total dose is 50 to 90 Gy, administered in 20 to 60 fractional doses of 2 to 3 Gy each. In some embodiments, the total dose of IR is at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 (or any range derivable therein). In some embodiments, the total dose is administered in fractional doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any range derivable therein). In some embodiments, at least, at most, or exactly 2, 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 (or any range derivable therein) fractional doses are administered. In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any range derivable therein) fractional doses are administered per day.In some embodiments, the sub-doses are administered at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any range derivable therein) times per week.

[0212] H. Surgery

[0213] Approximately 60% of people with cancer will undergo some type of surgery, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can be used in conjunction with other treatments, such as the treatments of embodiments of the application, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Resection of a tumor refers to the physical removal of at least a portion of a tumor. In addition to resection of a tumor, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs's surgery).

[0214] After resection of part or all of the cancerous cells, tissue, or tumor, a cavity can be formed in the body. Treatment can be accomplished by perfusion, direct injection, or local application of additional anti-cancer therapy to the area. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can also have a variety of dosages.

[0215] I. Other Agents

[0216] It is contemplated that other agents can be used in combination with certain aspects of embodiments of the application to increase 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, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Increasing 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 embodiments of the application to increase the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to increase the efficacy of embodiments of the application. Some examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, are also contemplated to be used in combination with certain aspects of embodiments of the application to increase the efficacy of the treatment.

[0217] V. Protein Compositions

[0218] As used herein, "protein," "peptide," or "polypeptide" refers to a molecule comprising at least five amino acid residues. As used herein, the term "wild type" refers to the endogenous form of a molecule that occurs naturally in an organism. In some embodiments, the wild type form of a protein or polypeptide is used, however, in many embodiments of the disclosure, a modified protein or polypeptide is used to generate an immune response. The above terms are used interchangeably. A "modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered relative to the wild type protein or polypeptide. In some embodiments, the modified / variant protein or polypeptide has at least one modified activity or function (a protein or polypeptide can have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide can be altered in one activity or function, but otherwise retains the wild type activity or function, e.g., immunogenicity.

[0219] Where a protein is specifically mentioned herein, it generally refers to a native (wild type) or recombinant (modified) protein, or optionally, a protein from which any signal sequence has been removed. A protein can be isolated directly from the organism of its natural source, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In some particular embodiments, there are isolated nucleic acid fragments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides (e.g., antibodies or fragments thereof). The term "recombinant" can be used in conjunction with the name of a polypeptide or a particular polypeptide, and this generally refers to a polypeptide that is produced from a nucleic acid molecule that has been manipulated in vitro or is a copy of such a molecule.

[0220] In some embodiments, the peptide, protein, or polypeptide (wild-type or modified), for example, comprising the peptide of SEQ ID NO:5, or the peptide or protein of the TCR embodiments of this disclosure of SEQ ID NO:2, 4, 6 to 11, 13, or 15, may be of a size including, but not limited to, at least, at most, or about 5, 6, 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, 35, 36, 37. , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525 The range includes 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any range that can be deduced from there. It is anticipated that the peptide can be mutated by truncation to make it shorter than its corresponding wild-type form, and that it can be altered by fusing or conjugating heterologous protein or peptide sequences with specific functions (e.g., for targeting or localization, for enhancing immunogenicity, for purification purposes, etc.).

[0221] The polypeptide, protein, or polynucleotide encoding such a polypeptide or protein as disclosed herein may comprise at least, at most, exactly, or about 1, 2, 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range deducible therefrom) or more variant amino acids or nucleic acids, or be substituted with at least, at most, SEQ ID NO: 1 to 15.

[0222] 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000

[0223] A contiguous amino acid or nucleic acid has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) sequence similarity, identity, or homology. In certain embodiments, the peptide or polypeptide is not naturally occurring, and / or in the combination of peptides or polypeptides.

[0224] In some embodiments, the protein or polypeptide or nucleic acid can comprise 1 to 2, 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, or 300,

[0225] amino acids or nucleic acids. In some embodiments, a peptide of the present disclosure comprises at least, at most, about, or exactly 1, 2, 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derivable therein) of the 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, or 270.

[0226] One continuous amino acid.

[0227] In some embodiments, the protein, polypeptide, or nucleic acid may comprise at least, at most, exactly, or about one of SEQ ID NO: 2, 4, 5 to 11, 13, or 15.

[0228] 1, 2, 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, or 270

[0229] A series of amino acids (or any range from which they can be derived).

[0230] In some embodiments, the polypeptide, protein, or nucleic acid may comprise at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or any range deducible therefrom) consecutive amino acids of the peptide or nucleic acid of SEQ ID NO: 1 to 15, said consecutive amino acids being in accordance with SEQ ID NO: 1 to 15. NO: 1 to 15 has at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range that can be derived therefrom) similarity, identity, or homology.

[0231] In some aspects, there is a polypeptide, nucleic acid (or nucleic acid molecule encoding such a polypeptide) that is at position 1, 2, 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, or 270,

[0232] beginning and comprising at least, at most, or exactly

[0233] 2, 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265 ， 266, 267, 268, 269, or 270

[0234] contiguous amino acids of the one (or any range derivable therein).

[0235] It is contemplated that from about 0.001 mg to about 10 mg of total polypeptide, peptide and / or protein per ml is present in the compositions of the disclosure. The concentration of protein in the composition can be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0236] The following is a discussion regarding altering the amino acid subunits of a protein to produce equivalent or even improved second generation variant polypeptides or peptides. For example, certain amino acids can replace other amino acids in a protein or polypeptide sequence without apparent loss of binding ability to interact with, for example, the structure of an antigen binding region of an antibody or a binding site on a substrate molecule. Since it is the interactive ability and nature of a protein that determines its functional activity, certain amino acid replacements can be made in a protein sequence and its corresponding DNA encoding sequence and yet produce a protein with similar or desirable properties. Thus, the present inventors contemplate that a variety of changes can be made in a DNA sequence of a gene encoding a protein without appreciable loss of its biological utility or activity.

[0237] The term "functionally equivalent codon" as used herein refers to a codon that encodes the same amino acid, for example, the six different codons for arginine. Also contemplated are "neutral substitutions" or "neutral mutations," which refer to changes in one or more codons that encode biologically equivalent amino acids.

[0238] Amino acid sequence variants of the present disclosure can be substitution, insertion, or deletion variants. Changes to the polypeptides of the present disclosure can affect 1, 2, 3, 4, 5, 6, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more (or any range derivable therein) noncontiguous or contiguous amino acids of the protein or polypeptide compared to the wild type. Variants can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90% (including all values and ranges therebetween) identical to any of the sequences provided or referred to herein. Variants can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitution amino acids.

[0239] It will also be appreciated that amino acid and nucleic acid sequences can comprise additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still be essentially identical to one set forth in a sequence disclosed herein, so long as the sequence meets the criteria set forth above, including maintaining biological protein activity where protein expression is concerned. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which can for example include multiple non-coding sequences flanking the 5' or 3' portion of a coding region.

[0240] Deletion variants typically lack one or more residues of the native or wild type protein. A single residue can be deleted or many contiguous amino acids can be deleted. A stop codon can be introduced (by substitution or insertion) into the coding nucleic acid sequence to produce a truncated protein.

[0241] Insertion mutants typically involve the addition of amino acid residues at a non- terminal point of the polypeptide. This can include the insertion of one or more amino acid residues. Terminal additions can also be made and can include fusion proteins that are multimers or concatemers of one or more of the peptides or polypeptides described or referred to herein.

[0242] Substitution variants typically comprise the exchange of one amino acid for another at one or more sites within a protein or polypeptide and can be designed to modulate one or more properties of the polypeptide with or without loss of other functions or properties. The substitution can be conservative, that is, one amino acid is replaced with one having similar chemical properties. "Conservative amino acid substitution" can involve the exchange of a member of one amino acid class for another member of the same class. Conservative substitutions are well known in the art and include, for example, the following alterations: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated through chemical peptide synthesis rather than through synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0243] Alternatively, the substitution can be "non-conservative," such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve the replacement of an amino acid residue by a chemically dissimilar amino acid residue, for example, the replacement of a polar or charged amino acid with a nonpolar or uncharged amino acid, or vice versa. Non-conservative substitutions can involve the replacement of a member of one amino acid class with a member from another class.

[0244] One skilled in the art can determine suitable variants of the polypeptides presented herein using well-known techniques. One skilled in the art can identify suitable regions of a molecule that can be altered without destroying activity by targeting regions that are not believed to be important to activity. One skilled in the art will also be able to identify amino acid residues and molecular moieties that are conserved among similar proteins or polypeptides. In other embodiments, conservative amino acid substitutions can be made to regions of biological activity or to regions that can be important to structure without significantly altering biological activity or adversely affecting the structure of the protein or polypeptide.

[0245] In making such changes, the hydropathic index of amino acids can be considered. The hydrophobic properties of a protein are calculated by assigning a value to each amino acid ("hydropathic index") and then averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydrophilic amino acid index in conferring interactive biologic functionality on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131 (1982)). It is recognized that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn influences the interaction of the protein or polypeptide with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). It is also known that certain amino acids can be substituted for other amino acids in certain situations where the following general principles are followed: amino acids with similar hydrophilicity values (the hydrophilicity of an amino acid in a peptide chain is understood to be influenced by the amino acids immediately adjacent to it, as well as by its own inherent hydrophilicity) can be substituted for one another; amino acids with similar hydrophilicity values and similar size can be substituted for one another; and / or amino acids with similar hydrophilicity values and similar charge range can be substituted for one another. In some embodiments, substitutions are made in accordance with the hydrophilicity index of amino acids, including substitutions of amino acids having a hydrophilicity within ±2 of the substituted amino acid. In some aspects of the application, those within ±1 are included, and in other aspects of the application, those within ±0.5 are included.

[0246] It is further understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In certain embodiments, where changes are made on the basis of similar hydrophilicity values, substitutions of amino acids include those in which relatively nonpolar amino acids such as alanine or valine are permutated for one another; leucine or isoleucine; asparagine or glutamine; serine or threonine; or lysine or arginine. In some cases, epitopes can also be identified from a primary amino acid sequence on the basis of hydrophilicity. These regions are also referred to as "epitope core regions." It is understood that an amino acid can be substituted for another amino acid that has similar hydrophilicity and still produce a biologically equivalent and immunologically equivalent protein.

[0247] Additionally, those of skill in the art could review the structure-function studies of identifying residues important to activity or structure in similar polypeptides or proteins. In light of such comparisons, one could predict the importance of amino acid residues in a protein that correspond to amino acid residues important to activity or structure in similar proteins. One of skill in the art could select a chemically similar amino acid substitution for such predicted important amino acid residues.

[0248] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence for structural relevance in similar proteins or polypeptides. Given such information, one skilled in the art can predict the arrangement of amino acid residues of a polypeptide relative to its three-dimensional structure. One skilled in the art can choose not to make alterations to amino acid residues predicted to be on the surface of the protein, as such residues can be involved in important interactions with other molecules. Furthermore, one skilled in the art can generate test variants comprising a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thereby gathering information from such routine experimentation, which can allow one skilled in the art to determine such amino acid positions where further substitutions, alone or in combination with other mutations, should be avoided. Various tools useful in determining secondary structure can be found on the World Wide Web at expasy.org / proteomics / protein_structure.

[0249] In some embodiments of the application, the following amino acid substitutions are made: (1) to decrease susceptibility to proteolysis, (2) to decrease susceptibility to oxidation, (3) to alter binding affinity for forming protein complexes, (4) to alter ligand or antigen binding affinity, and / or (5) to impart or modify other physicochemical or functional properties of the polypeptide. For example, a single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the naturally occurring sequence. The substitutions can be made in portions of the antibody outside of the domains that form intermolecular contacts. In some such embodiments, conservative amino acid substitutions that do not significantly alter the structural characteristics of the protein or polypeptide can be used (e.g., one or more alternative amino acids that do not disrupt the secondary structure characteristic of a naturally occurring antibody).

[0250] VI. Pharmaceutical Formulations

[0251] In some selected embodiments, it is contemplated that a Hormadl -derived peptide (e.g., SEQ ID NO: 5), a cell (e.g., a T cell) expressing a TCR as disclosed herein (e.g., any one of SEQ ID NOS: 1-4), or a protein comprising a TCR variable region of the disclosure can be administered to a subject to induce a therapeutic immune response against a cancer (e.g., a solid tumor expressing Hormadl) in the subject. A pharmaceutical composition for a subject can comprise a TCR disclosed herein, such as a soluble TCR (optionally linked to an imaging agent or a therapeutic agent) or a bispecific TCR, and a pharmaceutically acceptable carrier. If desired, the pharmaceutical composition can comprise an additional immunostimulatory compound or an anti-cancer agent.

[0252] The phrases "drug," "pharmaceutically acceptable," or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. "Pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2012, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the proteins (e.g., Hormadl peptides, soluble TCRs) or cells (e.g., T cells expressing TCRs) of the present disclosure, its use in the vaccine compositions or adoptive cell transfer therapies of the present application is contemplated. nd nd

[0253] "Therapeutic immune response" or "protective immune response" as used herein refers to the response of the immune system of a mammalian host to a cancer. A protective immune response can provide therapeutic effects for the treatment of cancer, such as reducing tumor size, increasing survival, etc.

[0254] A person of ordinary skill in the medical arts will appreciate that actual dosage amounts of the therapeutic compositions administered to an animal or human patient can be determined by physical and physiological factors (e.g., body weight, severity of disease, type of disease being treated, prior or concurrent therapeutic interventions, idiosyncratic response of the patient, and route of administration). In any case, the responsible practitioner will determine the concentration of active ingredient in the composition and appropriate dose for the individual subject.

[0255] The therapeutic compositions disclosed herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, transmucosally, intrapericardially, intraocularally, orally, topically, locally or by injection, infusion, continuous infusion, lavage and local perfusion. The therapeutic compositions can also be administered to a subject through a catheter, in a lipid composition or by other methods or any combination of the above methods, as known to one of ordinary skill in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 22 ndEd., Pharmaceutical press, 2012, incorporated herein by reference).

[0256] While any suitable carrier known to those of ordinary skill in the art can be used in the pharmaceutical compositions of the present application, the type of carrier will vary depending on the mode of administration. For parenteral administration (e.g., intravenous, intratumoral, or subcutaneous injection), the carrier can comprise water, saline, alcohol, fat, wax, or a buffer. In some embodiments, biodegradable microspheres (e.g., polylactic galactide) can also be used as carriers. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patents 4,897,268 and 5,075,109.

[0257] In some embodiments, the vaccine compositions can be administered by microstructured transdermal delivery or ballistic microparticle delivery. Microstructures as carriers of vaccine formulations are desirable constructs for vaccine applications, and are widely known in the art (e.g., U.S. Patents 5,797,898, 5,770,219, and 5,783,208, and U.S. Patent Application 2005 / 0065463). Microstructures or ballistic particles used as support substrates for TCRs (e.g., soluble TCRs disclosed herein) can comprise biodegradable materials and non-biodegradable materials, and such support substrates can comprise synthetic polymers, silica, lipids, carbohydrates, proteins, lectins, ionic agents, cross-linking agents, and other microstructure components available in the art. Protocols and reagents for immobilizing the peptides of the present application to support substrates comprised of such materials are widely commercially available.

[0258] In other embodiments, the vaccine compositions comprise an immobilized or encapsulated TCR or soluble TCR disclosed herein and a support substrate. Support substrates can include, but are not limited to, lipid microspheres, lipid nanoparticles, ethosomes, liposomes, niosomes, phospholipids, sphingosomes, surfactants, transferosomes, emulsions, or combinations thereof. The formation and use of liposomes and other lipid nanocarrier formulations and lipid microcarrier formulations are generally known to those of ordinary skill in the art, and the use of liposomes, microparticles, nanocapsules, and the like has found wide application in the delivery of therapeutic agents (e.g., U.S. Patent 5,741,516, specifically incorporated herein in its entirety by reference). Many methods are known for liposomes and liposome-like formulations as potential drug carriers, which include peptide encapsulation, and which can be used in various embodiments (U.S. Patents 5567434, 5552157, 5565213, 5738868, and 5795587).

[0259] In any event, the compositions can include antioxidants to retard oxidation of one or more components. Additionally, prevention of the action of microorganisms can be achieved by preservatives, such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0260] The compositions must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, such as less than 0.5 ng / mg of protein.

[0261] A. Combination Therapy

[0262] In certain embodiments, the compositions and methods of embodiments of the application comprising a population of antigen-specific cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, or gamma-delta T cells), NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), or induced pluripotent stem (iPS) cells) can be administered to a mammalian subject (e.g., a human) in combination with at least one additional therapy. The additional therapy can be radiation therapy, surgery (e.g., primary surgery, tumor resection, lumpectomy, or mastectomy), chemotherapy, conditioning chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy can be in the form of an adjuvant therapy or a neoadjuvant therapy.

[0263] In some embodiments, the additional therapy is administration of a small molecule enzymatic inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of one or more side effect limiting agents (e.g., an agent that reduces the occurrence and / or severity of side effects of a therapy, such as an anti-nausea agent, etc.). 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 chemotherapy, such as dacarbazine or temozolomide. The additional therapy can be one or more chemotherapeutic agents known in the art.

[0264] T cell therapy or adoptive cell transfer therapy can be administered prior to, during, after, or in various combinations relative to another cancer treatment, such as an immune checkpoint therapy or conditioning chemotherapy. Administration can be within a simultaneous to minutes to days to weeks interval. In some embodiments where the T cell therapy is provided to the patient separately from the additional therapeutic agent, one will generally ensure that a substantial period of time does not expire between each delivery time, such that the two compounds are still able to exert a beneficial combined effect on the patient. In such cases, it is contemplated that the antibody therapy and the anti-cancer therapy can be provided to the patient within about 12 to 24 or 72 hours of each other, and more particularly, within about 6 to 12 hours of each other. In some cases, it can be desirable to extend the time period of the treatment significantly, where the interval between respective administrations is extended from days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8).

[0265] A variety of combinations can be used. For the following examples, the antigen-specific T cell therapy, peptide, or TCR is "A" and the anti-cancer therapy is "B":

[0266] A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B

[0267] B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A

[0268] B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0269] Administration of any compound or therapy of embodiments of the present application to a patient will follow general protocols for administration of such compounds, taking into account the toxicity, if any, of the agents. Thus, in some embodiments, there is a step of monitoring toxicity attributable to the combination therapy.

[0270] VII. Examples

[0271] The following examples are included to demonstrate some preferred embodiments of the application. Those of skill in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the application, and, thus, can be considered to be preferred modes of practicing the application. However, various modifications can be made without departing from the spirit and scope of the application, as defined by the claims.

[0272] Example 1

[0273] Hormad1-specific T-cell receptor redirects T cells targeting tumor cells.

[0274] To further explore the potential of the Hormad1 T cell epitope as a therapeutic target for clinical immunotherapy, the full-length Hormad1-56TCR α and β chains were inserted into the retroviral vector pMSGV3, and the recombinant retroviral vector was subsequently used to infect PBMCs (…). FIG. 3 Empty retroviral vectors were used as controls. Following infection, a CD8+ / tetrameric+ population was observed using FCM. Highly purified TCR-T cells were generated after tetramer-guided sorting and amplification. Although Hormad1 overexpression was observed in tumor tissues of approximately 50% of non-small cell lung cancer (NSCLC) patients, elevated Hormad1 expression was not observed in healthy tissues (Hormad1 is not expressed in healthy tissues except the testes), and high Hormad1 expression was associated with elevated mutational burden in the lung adenocarcinoma patient population (Nichols et al., 2018). It remains unclear whether this protein could serve as a target for immunotherapy.

[0275] T cells transduced via TCR were observed to specifically recognize Hormad1 peptide pulses with high affinity and lyse HLA-A2+ Hormad1-expressing tumor cell lines, but not Hormad1- or HLA-A2-, or normal cells (Fig. 4). T cells transduced via a Hormad1-specific TCR recognized these solid tumor cells but not control tumor cells (Fig. 4). These results suggest that Hormad1-derived peptides are expressed on tumor cells in the context of HLA-A2 molecules, and that Hormad1-TCR-transduced T cells could be used for cancer immunotherapy.

[0276] Hormad1-56 TCR-T functional assay

[0277] To further explore the function of Hormad1-56 TCR-T cells, cytokine production was detected by intracellular staining assays (Pala Pietro, et al., J Immunol Methods. 2010; 243(1-2):107-124). Hormad1-56 TCR-T cells were co-cultured with several tumor cell lines. It was observed that when co-cultured with HLA-A2+ Hormad1-expressing tumor cell lines (but not antigen-negative cells or control tumor cell lines), the levels of CD137, CD69, TNF-α, and IFN-γ expressed by TCR-T cells were significantly increased. FIG. 5 ).

[0278] TCR sequences were derived from the parental Hormadl-56 CTL cell line A12. It revealed that the Hormadl-56 TCR (hereinafter referred to as Hormadl-TCR) is composed of TRAV4*01F, TRBV13*01F and TRAV4*01F, TRBV13*01F 2-subfamily sequences (Figure 6).

[0279] Example 2

[0280] Materials and Methods

[0281] Healthy donor PBMC samples

[0282] The study was approved by the Institutional Review Board of The University of Texas M.D. Anderson Cancer Center. Informed consent was obtained in accordance with the Declaration of Helsinki prior to collection of healthy donor PBMC samples. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by leukapheresis.

[0283] Cell lines

[0284] T2 hybridoma cells, lung cancer cell lines H1395, H522, H1299, H1299-A2, H1355, H1755, DFC1032, K562-A2, K562-A2-eGFP, K562-A2-Hormadl, H522-eGFP, H522-Hormadl were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 1 x Glutamax, 50 μΜ β-mercaptoethanol, 1 mM sodium pyruvate, 100 U / mL penicillin + 100 μg / mL streptomycin, and 10 μg / mL gentamicin (all from Invitrogen, Carlsbad, CA) at 37 °C in air with 5% CO2. Normal lung cell line HSAEC2-KT was cultured in serum-free Small Airway Epithelial Cell Growth Medium (PromoCell, Heidelberg, Germany).

[0285] Tumor and immune cell subpopulation isolation

[0286] CD25-T cells were isolated by magnetic cell separation (MACS, Miltenyi Biotec, Auburn, CA) and purity was confirmed by flow. This procedure yielded >90% pure CD25-T cells.

[0287] Reagents

[0288] Mouse anti-human antibodies against CD3, CD4, CD8, CD69, CD137, IFN-g, TFN-b were obtained from Biolegend, San Diego, CA. All peptides were synthesized to greater than 90% purity by Genscript, Piscataway, NJ and dissolved in dimethyl sulfoxide (Sigma-Aldrich). PE-conjugated tetramers were synthesized by the Immune Monitoring Center of Fred Hutchinson Cancer Research Center, Seattle, WA.

[0289] PCR

[0290] Total RNA was extracted from T cells using the RNeasy kit (Qiagen). Approximately 3 pg of total RNA was reverse transcribed into cDNA using the RACE 5’ / 3’ kit (ClonTech). PCR was performed using the Phusion® High-Fidelity 2x Master Mix kit (NEB) using the following conditions: 98°C for 2 minutes, followed by 98°C for 15 seconds, 63°C for 30 seconds, 72°C for 45 seconds, for 40 cycles on a Bio-Rad PCR system. PCR products were cloned into the pRACE vector using the In Fusion clone kit (ClonTech) and subsequently sequenced using the Direct Cycle Sequencing kit (Thermo).

[0291] Flow cytometry

[0292] ​​​For intracellular staining, cells were fixed and permeabilized using the Fixation / Permeabilization Kit (eBioscience) according to the manufacturer's instructions. Cells were then stained with mouse anti-human flow antibodies (as described above; 00114) for 30 min at 4°C. After two washes, samples were acquired on a FACS Calibur (BD Biosciences) and analyzed using CellQuest Pro (BD Biosciences) or FlowJo (Tree Star, Inc., Ashland, OR) software. Intracellular cytokine staining was performed as previously described (Weng et al., 2016b). For tetramer staining, PE-conjugated Hormadl tetramer and APC-Cy7-conjugated mouse anti-human CD8 antibody were mixed with cells in a 50 μΐ volume for 30 min at room temperature, washed twice, and analyzed by flow cytometry.

[0293] Generation of Hormad1-56 peptide-specific CTL lines

[0294] Mature DCs derived from HLA-A0201+ healthy donors were pulsed with Hormadl-56 peptide (YLDDLCVKI; SEQ ID NO: 5) and stimulated with autologous CD25-T cells. After two rounds of stimulation, Hormadl-56 specific T cell lines were detected and sorted with the corresponding Hormadl-56 tetramer and anti-CD8 antibody. CD8+ / tetramer+ T cells were expanded with a rapid expansion protocol (REP) and stained with anti-CD8 antibody and tetramer to determine the purity of Hormadl-56 specific T cells.

[0295] Production of Hormad1-specific TCR-T cells by retrovirus

[0296] The complete TCRaP sequence of the Hormadl T cell line was obtained by 5-RACE RT-PCR and codon-optimized. The constant regions of the a and β chains were cysteine mutated; the TCRaP chain was linked with furin and P2A and cloned into a vector that produces retrovirus. Retrovirus containing the TCR was produced in 293T cells, filtered, concentrated, and stored at -80°C. HLA-A2+ healthy donor T cells were activated with OKT3 antibody and IL-2 for 72 h and transduced with the retrovirus at 32°C for 2 h followed by overnight incubation with centrifugation at 2000 g. Expression of the antigen-specific TCR was analyzed by tetramer staining after 48 h. Tetramer-positive T cells were sorted by flow cytometry as previously described and further expanded by REP for additional functional assays (Pollack et al., 2014).

[0297] Cytotoxicity assay

[0298] T2 cells were pulsed with decreasing concentrations of peptide (10 μg / ml to 10 pg / ml) and used as targets in standard 4 hour Cr51 release cytotoxicity assays. Tumor cell lines (2 x 10 3 Cells were incubated at 37°C for 4 hours and target cell lysis was determined by Cr51 release assay. All assays were performed in triplicate wells and repeated at least twice.

[0299] Statistical analysis

[0300] Student's t test was used to compare multiple experimental groups. P values < 0.05 were considered statistically significant. Mean and standard deviation are shown unless otherwise indicated.

[0301] ***

[0302] In accordance with the present disclosure, all methods herein disclosed and claimed can be made and executed without undue experimentation. While compositions and methods of the present application have been described in terms of some preferred embodiments, it will be apparent to those of ordinary skill in the art that many modifications, substitutions, and changes can be made to the methods described herein and to the steps or order of steps used therein without departing from the concept, spirit and scope of the application. More specifically, it will be apparent that certain agents which are both chemically and physiologically related can be substituted for the agents described herein and be equally effective. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the application as defined by the appended claims.

[0303] REFERENCES BACKGROUND SUMMARY FIGS. 1A-1D FIG. 1A FIG. 1B FIG. 1C FIG. 1D FIG. 2 FIG. 3 FIG. 5 FIGS. 6A-6B FIG. 6A FIG. 6B DETAILED DESCRIPTION FIGS. 6A-6B FIGS. 6A-6B FIGS. 6A-6B FIGS. 6A-6B FIGS. 6A-6B FIG. 3 Hormad1-56 TCR-T functional assay FIG. 5 Healthy donor PBMC samples Cell lines Tumor and immune cell subpopulation isolation Reagents PCR

[0304] The following references are specifically incorporated herein by reference for their exemplary procedures or other details that supplement those set forth herein.

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SEQUENCE LISTING <110> Board of Regents, The University of Texas System <120> HLA-restricted HORMAD1 T cell receptors and uses thereof <130> UTSC.P1238WO <140> <141> 2020-11-05 <150> 62 / 930,892 <151> 2019-11-05 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 804 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 1 atgaggcaag tggcgagagt gatcgtgttc ctgaccctga gtactttgag ccttgctaag 60 accacccagc ccatctccat ggactcatat gaaggacaag aagtgaacat aacctgtagc 120 cacaacaaca ttgctacaaa tgattatatc acgtggtacc aacagtttcc cagccaagga 180 ccacgattta ttattcaagg atacaagaca aaagttacaa acgaagtggc ctccctgttt 240 atccctgccg acagaaagtc cagcactctg agcctgcccc gggtttccct gagcgacact 300 gctgtgtact actgcctcgt gggtgcgcgg ggaactgctc tgatctttgg gaagggaacc 360 accttatcag tgagttccaa tatccagaac cctgaccctg ccgtgtacca gctgagagac 420 tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca aacaaatgtg 480 tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga catgaggtct 540 atggacttca agagcaacag 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Tyr Gin Leu Arg Asp Ser Lys Ser Ser 130 135 140 Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gin Thr Asn Val 145 150 155 160 Ser Gin Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu 165 170 175 Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser 180 185 190 Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile 195 200 205 Pro Gin Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu 210 215 220 Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gin Asn 225 230 235 240 Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe 245 250 255 Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 3 <211> 963 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 3 atgcttagtc ctgacctgcc tgactctgcc tggaacacca ggctcctctg ccatgtcatg 60 ctttgtctcc tgggagcagg ttcagtggct gctggagtca tccagtcccc aagacatctg 120 atcaaagaaa agagggaaac agccactctg aaatgctatc ctatccctag acacgacact 180 gtctactggt accagcaggg tccaggtcag gacccccagt tcctcatttc gttttatgaa 240 aagatgcaga gcgataaagg aagcatccct gatcgattct cagctcaaca gttcagtgac 300 tatcattctg aactgaacat gagctccttg gagctggggg actcagccct gtacttctgt 360 gccagcagcc ctacgggaca gggttcgtac gagcagtact tcgggccggg caccaggctc 420 acggtcacag aggacctgaa aaacgtgttc ccacccgagg tcgctgtgtt tgagccatca 480 gaagcagaga tctcccacac ccaaaaggcc acactggtgt gcctggccac aggcttcttc 540 cctgaccacg tggagctgag ctggtgggtg aatgggaagg aggtgcacag tggggtcagc 600 acggacccgc agcccctcaa ggagcagccc gccctcaatg actccagata ctgcctgagc 660 agccgcctga gggtctcggc caccttctgg cagaaccccc gcaaccactt ccgctgtcaa 720 gtccagttct acgggctctc ggagaatgac gagtggaccc aggatagggc caaacccgtc 780 acccagatcg tcagcgccga ggcctggggt agagcagact gtggctttac ctcggtgtcc 840 taccagcaag gggtcctgtc tgccaccatc ctctatgaga tcctgctagg gaaggccacc 900 ctgtatgctg tgctggtcag cgcccttgtg ttgatggcca tggtcaagag aaaggatttc 960 taa 963 <210> 4 <211> 320 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 4 Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 Cys His Val Met Leu Cys Leu Leu Gly Ala Gly Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Pro Thr Gly Gln Gly 115 120 125 Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr Glu 130 135 140 Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser 145 150 155 160 Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala 165 170 175 Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 180 185 190 Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu 195 200 205 Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg 210 215 220 Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln 225 230 235 240 Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg 245 250 255 Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala 260 265 270 Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser Ala 275 280 285 Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val 290 295 300 Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Phe 305 310 315 320 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 5 Tyr Leu Asp Asp Leu Cys Val Lys lie 1 5 <210> 6 <211> 7 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 6 Asn lie Ala Thr Asn Asp Tyr 1 5 <210> 7 <211> 5 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 7 Gly Tyr Lys Thr Lys 1 5 <210> 8 <211> 11 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 8 Leu Val Gly Ala Arg Gly Thr Ala Leu lie Phe 1 5 10 <210> 9 <211> 5 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 9 Pro Arg His Asp Thr 1 5 <210> 10 <211> 6 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 10 Phe Tyr Glu Lys Met Gin 1 5 <210> 11 <211> 13 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 11 Ala Ser Ser Pro Thr Gly Gin Gly Ser Tyr Glu Gin Tyr 1 5 10 <210> 12 <211> 330 <212> DNA <213> artificial sequence <220> <223> synthetic oligonucleotide <400> 12 cttgctaaga ccacccagcc catctccatg gactcatatg aaggacaaga agtgaacata 60 acctgtagcc acaacaacat tgctacaaat gattatatca cgtggtacca acagtttccc 120 agccaaggac cacgatttat tattcaagga tacaagacaa aagttacaaa cgaagtggcc 180 tccctgttta tccctgccga cagaaagtcc agcactctga gcctgccccg ggtttccctg 240 agcgacactg ctgtgtacta ctgcctcgtg ggtgcgcggg gaactgctct gatctttggg 300 aagggaacca ccttatcagt gagttccaat 330 <210> 13 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 13 Leu Ala Lys Thr Thr Gin Pro lie Ser Met Asp Ser Tyr Glu Gly Gin 1 5 10 15 Glu Val Asn lie Thr Cys Ser His Asn Asn lie Ala Thr Asn Asp Tyr 20 25 30 lie Thr Trp Tyr Gin Gin Phe Pro Ser Gin Gly Pro Arg Phe lie lie 35 40 45 Gln Gly Tyr Lys Thr Lys Val Thr Asn Glu Val Ala Ser Leu Phe lie 50 55 60 Pro Ala Asp Arg Lys Ser Ser Thr Leu Ser Leu Pro Arg Val Ser Leu 65 70 75 80 Ser Asp Thr Ala Val Tyr Tyr Cys Leu Val Gly Ala Arg Gly Thr Ala 85 90 95 Leu lie Phe Gly Lys Gly Thr Thr Leu Ser Val Ser Ser Asn 100 105 110 <210> 14 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 14 gctgctggag tcatccagtc cccaagacat ctgatcaaag aaaagaggga aacagccact 60 gctgctggag tcatccagtc cccaagacat ctgatcaaag aaaagaggga aacagccact 60 ctgaaatgct atcctatccc tagacacgac actgtctact ggtaccagca gggtccaggt 120 ctgaaatgct atcctatccc tagacacgac actgtctact ggtaccagca gggtccaggt 120 caggaccccc agttcctcat ttcgttttat gaaaagatgc agagcgataa aggaagcatc 180 caggaccccc agttcctcat ttcgttttat gaaaagatgc agagcgataa aggaagcatc 180 cctgatcgat tctcagctca acagttcagt gactatcatt ctgaactgaa catgagctcc 240 cctgatcgat tctcagctca acagttcagt gactatcatt ctgaactgaa catgagctcc 240 ttggagctgg gggactcagc cctgtacttc tgtgccagca gccctacggg acagggttcg 300 ttggagctgg gggactcagc cctgtacttc tgtgccagca gccctacggg acagggttcg 300 tacgagcagt acttcgggcc gggcaccagg ctcacggtca ca 342 tacgagcagt acttcgggcc gggcaccagg ctcacggtca ca 342 <210> 15<210> 15 <211> 114<211> 114 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220><220> <223> 合成多肽<223> Synthetic polypeptide <400> 15 <400> 15 Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg 1 5 10 15 1 5 10 15 Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val 20 25 30 20 25 30 Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser 35 40 45 35 40 45 Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe 50 55 60 50 55 60 Ser Ala Gin Gin Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser 65 70 75 80 Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Pro Thr 85 90 95 Gly Gin Gly Ser Tyr Glu Gin Tyr Phe Gly Pro Gly Thr Arg Leu Thr 100 105 110 Val Thr

Claims

1. An engineered T-cell receptor (TCR) having antigen specificity for binding to HLA-A2 SEQ ID NO:5, wherein the TCR comprises an α-chain variable region and a β-chain variable region, the α-chain variable region and the β-chain variable region comprising: (i) TCRαCDR1 consisting of the amino acid sequence of SEQ ID NO:6; (ii) TCRαCDR2 consisting of the amino acid sequence of SEQ ID NO:7; (iii) TCRαCDR3 consisting of the amino acid sequence of SEQ ID NO:8; (iv) TCRβCDR1, which consists of the amino acid sequence of SEQ ID NO:9; (v) TCRβCDR2, consisting of the amino acid sequence of SEQ ID NO:10; and (vi) TCRβCDR3, which consists of the amino acid sequence of SEQ ID NO:

11.

2. The engineered TCR of claim 1, wherein the engineered TCR comprises: (i) an α-chain variable region having a sequence having at least 90% sequence identity with SEQ ID NO: 13 or 2; and / or (ii) β chain variable region having a sequence having at least 90% sequence identity with SEQ ID NO:15 or 4.

3. The engineered TCR of claim 2, wherein the engineered TCR comprises: (i) the α-chain variable region having the amino acid sequence of SEQ ID NO: 13 or 2; and / or (ii) β-chain variable region having an amino acid sequence of SEQ ID NO:15 or 4.

4. The engineered TCR according to any one of claims 1 to 3, wherein the α chain and the β chain are each modified to include cysteine ​​residues in their constant regions, such that disulfide bonds are formed, wherein each cysteine ​​residue is not naturally occurring.

5. The engineered TCR according to any one of claims 1 to 4, wherein the engineered TCR comprises modifications and / or inlays.

6. The engineered TCR according to any one of claims 1 to 5, wherein the engineered TCR is further defined as a single-chain TCR (scTCR), wherein the α chain and the β chain are covalently connected by a flexible joint.

7. The engineered TCR according to any one of claims 1 to 6, wherein the engineered TCR comprises a bispecific TCR.

8. The engineered TCR of claim 7, wherein the bispecific TCR comprises scFv that targets or selectively binds to CD3.

9. A multivalent TCR complex comprising a plurality of engineered TCRs as described in any one of claims 1 to 8.

10. The multivalent TCR complex of claim 9, wherein the multivalent TCR comprises 2, 3, 4 or more TCRs associated with each other.

11. The multivalent TCR complex of claim 9, wherein the multivalent TCR is present in a lipid bilayer, in liposomes, or attached to nanoparticles.

12. The multivalent TCR complex of claim 9, wherein the engineered TCRs are associated with each other through linker molecules or non-naturally occurring disulfide bonds.

13. One or more nucleic acids comprising a nucleotide sequence encoding an engineered TCR according to any one of claims 1 to 8.

14. The nucleic acid of claim 13, wherein the nucleic acid comprises cDNA encoding the engineered TCR.

15. An expression vector comprising the nucleic acid of claim 13 or 14.

16. The expression vector of claim 15, wherein the expression vector comprises sequences encoding the TCRα and TCRβ genes.

17. The expression vector of claim 15 or 16, wherein the nucleotide sequence encoding the engineered TCR is under the control of a promoter.

18. The expression vector of any one of claims 15 to 17, wherein the expression vector is a viral vector.

19. The expression vector of claim 18, wherein the viral vector is a retroviral vector or a lentiviral vector.

20. A host cell engineered to express an engineered TCR according to any one of claims 1 to 8, wherein the host cell comprises the nucleic acid according to claim 13 or 14 or the expression vector according to any one of claims 15 to 19.

21. The host cell of claim 20, wherein the host cell is a T cell, NK cell, constant NK cell, NKT cell, mesenchymal stem cell (MSC) or induced pluripotent stem cell (iPS) cell.

22. The host cell of claim 20, wherein the host cell is an immune cell.

23. The host cell of any one of claims 20 to 22, wherein the host cell is separated from the umbilical cord.

24. The host cell of claim 21, wherein the T cell is a CD8+ T cell, a CD4+ T cell, or a γδ T cell.

25. The host cell of claim 21, wherein the T cell is a regulatory T cell (Treg).

26. The host cell of any one of claims 20 to 25, wherein the cell is autologous.

27. The host cell of any one of claims 20 to 25, wherein the cell is an allogeneic cell.

28. A method for engineering host cells according to any one of claims 22 to 23 and 26 to 27, comprising contacting the immune cells with the nucleic acid of claim 13 or 14 or the expression vector of any one of claims 15 to 19.

29. The method of claim 28, wherein the immune cell is a T cell or a peripheral blood lymphocyte.

30. The method of claim 28 or 29, wherein the contact is further defined as transfection or transduction.

31. The method of claim 30, wherein transfection comprises electroporating the immune cells with RNA encoding the TCR of any one of claims 1 to 7.

32. The method of claim 30, further comprising generating a viral supernatant from the expression vector of claim 18 prior to transducing the immune cells.

33. The method of any one of claims 28 to 32, wherein the immune cells are stimulated lymphocytes.

34. The method of claim 33, wherein the stimulated lymphocytes are human lymphocytes.

35. The method of claim 33, wherein the stimulation comprises contacting the immune cells with OKT3 and / or IL-2 or incubating the immune cells in OKT3 and / or IL-2.

36. The method of any one of claims 28 to 35, further comprising sorting the immune cells to isolate TCR-engineered T cells.

37. The method of claim 36, further comprising T-cell cloning via serial dilution.

38. The method of claim 37, further comprising amplifying the T cell clone using a rapid amplification protocol.

39. Use of the host cell of any one of claims 20 to 27 for the preparation of a medicament for treating cancer in a mammalian subject, comprising administering an effective amount of the host cell to the mammalian subject in which such treatment is desired, wherein the cancer expresses the amino acid sequence of Hormad1 or SEQ ID NO:

5.

40. The use according to claim 39, wherein the TCR-engineered cells are T cells or peripheral blood lymphocytes.

41. The use according to claim 40, wherein the T cell is a CD8+ T cell, NK T cell, iNKT cell, CD4+ T cell, or Treg.

42. The use according to any one of claims 39 to 41, wherein the cancer is breast cancer, lung cancer, esophageal cancer, bone cancer, endometrial cancer, hematopoietic system cancer or lymphoma, gastrointestinal cancer, ovarian cancer, skin cancer, neuroblastoma, testicular cancer, thymoma, bladder cancer, uterine cancer, melanoma, sarcoma, cervical cancer, or head and neck cancer.

43. The use according to any one of claims 39 to 42, wherein the cancer is a solid tumor.

44. The use according to any one of claims 39 to 43, wherein the mammalian object is a human being.

45. The use according to any one of claims 39 to 44, wherein the TCR-engineered cells are autologous or allogeneic to the mammalian subject.

46. ​​The use according to any one of claims 39 to 45, further comprising lymphocyte depletion of the mammalian subject prior to administration of Hormad1-specific T cells.

47. The use of claim 46, wherein the lymphocyte depletion comprises the administration of cyclophosphamide and / or fludarabine.

48. The use according to any one of claims 39 to 47, further comprising administering a second anticancer treatment to the mammalian subject.

49. The use according to claim 48, wherein the second anticancer treatment is chemotherapy, immunotherapy, surgery, radiotherapy or biotherapy.

50. The use according to any one of claims 39 to 49, wherein the TCR-engineered cells and / or at least the second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.

51. The use according to any one of claims 39 to 50, wherein the object is identified as having or diagnosed as having cancer cells overexpressing Hormad1.

52. One or more nucleic acids comprising cDNA encoding the TCRα-strand variable region and the TCRβ-strand variable region of claim 1.

53. An RNA molecule encoding both the TCRα chain variable region and the TCRβ chain variable region of claim 1.

54. T cells comprising the nucleic acid of claim 52 or the RNA molecule of claim 53.

55. An in vitro or ex vivo method for activating or expanding Hormad1-specific T cells, wherein the Hormad1-specific T cells are antigen-specific to SEQ ID NO:5 binding to HLA-A2, the method comprising: (a) Obtaining a starting cell population from a mammalian object, wherein the starting cell population comprises T cells; (b) In vitro contacting the starting cell population with the nucleic acid of claim 13 or 14 or the expression vector of any one of claims 15 to 19, and activating Hormad1-specific T cells, stimulating the proliferation of Hormad1-specific T cells, and / or expanding Hormad1-specific T cells in the starting population; and (c) Isolate the Hormad1-specific T cells.

56. The method of claim 55, wherein the starting cell population is obtained from a blood sample of a mammalian subject.

57. The method of claim 55, wherein contacting the initiating cell population in vitro further comprises co-culturing the initiating T cell population with antigen-presenting cells (APCs), wherein the APCs are capable of presenting an amino acid sequence comprising SEQ ID NO:5 on their surface.

58. The method of claim 57, wherein the APC is a dendritic cell.

59. The method of claim 58, wherein the dendritic cells are autologous dendritic cells obtained from the mammalian object.

60. The method of claim 55, wherein the contact is further defined as co-culturing the initiating T cell population with artificial antigen-presenting cells (aAPC).

61. The method of claim 60, wherein the artificial antigen-presenting cell (aAPC) comprises or is composed of the following: Poly(lactide-co-glycolic acid) (PLGA), K562 cells, paramagnetic beads coated with CD3 and CD28 agonist antibodies, beads or microparticles coupled with HLA-dimer and anti-CD28, or nano-sized aAPCs (nano-aAPCs) with a diameter of less than 100 nm.

62. The method of any one of claims 58 to 61, wherein the T cells are CD8+ cells. + T cells or CD4 + T cells.

63. The method of any one of claims 58 to 62, wherein the T cell is a cytotoxic T lymphocyte (CTL).

64. The method of any one of claims 58 to 63, wherein the starting cell population comprises or is composed of peripheral blood mononuclear cells (PBMCs).

65. The method of claim 64, wherein the method further comprises isolating or purifying the T cells from the peripheral blood mononuclear cells (PBMCs).

66. The method of any one of claims 58 to 65, wherein the mammalian object is a human being.

67. The method of any one of claims 58 to 66, wherein the method further comprises re-infusing or administering activated or expanded Hormad1-specific T cells to the subject.

68. The activated or expanded Hormad1-specific T cells according to any one of claims 58 to 67.

69. A pharmaceutical composition comprising activated or expanded Hormad1-specific T cells according to any one of claims 58 to 68.

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