HLA-restricted HORMAD1 T cell receptor and its use

Abstract

To further improve clinical outcomes, this provides a novel target for adoptive T-cell therapy approaches. [Solution] A T cell receptor (TCR) and a TCR variable region are provided that can selectively bind to the Hormad1 peptide / MHC complex. The TCR can be used in a variety of therapies, such as autologous Hormad1-TCR adoptive T cell therapy for treating cancers such as solid tumors that express Hormad1. A method for expanding and proliferating the associated T cell population is provided.

Description

[Technical Field]

[0001] This application claims priority under U.S. Provisional Patent Application No. 62 / 930,892, filed on 5 November 2019, which is incorporated herein by reference in its entirety.

[0002] 1. Field of Invention This invention broadly relates to the fields of immunology and medicine. More specifically, it relates to antigen peptides and recombinant T cell receptors (TCRs). In some embodiments, TCRs can be used to treat cancer. [Background technology]

[0003] 2. Explanation of related technologies While T-cell-based therapies have shown promise in treating various cancers, relapse after immunotherapy or chemotherapy remains a significant clinical problem. Aggressive B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) are often responsive to a combination of chemotherapy and anti-CD20 monoclonal antibodies (Plosker and Figgitt, 2003), but approximately one-third of patients experience repeated relapses and ultimately die from the disease (Chao MP, 2013). Recent studies using CD19-targeted chimeric antigen receptor (CAR)-modified T-cell therapy have achieved complete remission (CR) rates of 60–90% 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). Furthermore, some of these patients experienced long-term remission, supporting the idea that adoptive T-cell therapy can be an effective treatment and, in some cases, even curative. Nevertheless, more than half of the treated patients relapsed after CD19 CAR T-cell therapy, primarily due to the loss of CD19 expression on the tumor (Sotillo et al., 2015; Topp et al., 2014; Neelapu et al., 2017). Clearly, novel targets for adoptive T-cell therapy approaches are needed to further improve clinical outcomes. [Overview of the project]

[0004] This disclosure overcomes the limitations of the prior art in several respects by providing a Hormad1 peptide (e.g., SEQ ID NO:5) recognized by HLA-A2 and a T cell receptor (TCR) capable of binding to the Hormad1 peptide / MHC I complex. This peptide and TCR may be used, for example, in adoptive T cell therapy or soluble T cell therapy for treating cancer.

[0005] One aspect of this disclosure relates to isolated Hormad1 peptides of 35 amino acids or less in length, comprising SEQ ID NO:5, an amino acid sequence having at least 85% sequence identity with SEQ ID NO:5, an amino acid sequence containing at least six consecutive amino acids of SEQ ID NO:5, or an amino acid sequence having only one substitution mutation with respect to SEQ ID NO:5.

[0006] 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 with SEQ ID NO: 5. In some embodiments, the peptide comprises an amino acid sequence comprising at least 5, 6, 7, 8, or 9 consecutive amino acids of SEQ ID NO: 5.

[0007] The peptide may 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 may be further defined as an immunogenic peptide and / or a peptide that can induce cytotoxic T lymphocytes (CTLs) and selectively binds to HLA-A2. The term immunogenic may refer to producing an immune response, such as a protective immune response. In some embodiments, the peptide is modified. In some embodiments, the modification involves binding to a molecule. The molecule may be an antibody, lipid, adjuvant, or detection portion (tag).

[0008] Another aspect of this disclosure relates to pharmaceutical compositions comprising isolated peptides (e.g., SEQ ID NO: 5) and pharmaceutical carriers as described herein or above. The pharmaceutical compositions may be formulated for parenteral administration, intravenous injection, intramuscular injection, or subcutaneous injection. In some embodiments, the pharmaceutical compositions comprise liposomes, lipid-containing nanoparticles, or lipid-based carriers. In some embodiments, the pharmaceutical preparations are formulated for injection. In some embodiments, the pharmaceutical preparations are formulated for inhalation. The pharmaceutical preparations may comprise or consist of nasal sprays.

[0009] Another aspect of this disclosure relates to isolated nucleic acids encoding Hormad1-derived peptides (e.g., SEQ ID NO: 5) as described herein or above.

[0010] Another aspect of this disclosure relates to vectors containing nucleic acids as described herein or above.

[0011] Isolated host cells comprising the nucleic acids, peptides, TCRs, and vectors of the present disclosure are also provided.

[0012] A further aspect relates to a method of making a cell, comprising the step of introducing a nucleic acid or vector of the present disclosure into a cell.

[0013] Another aspect of this disclosure relates to methods for stimulating an immune response in a mammalian subject, comprising the step of administering an effective amount of a peptide described herein or above (e.g., SEQ ID NO: 5) to the subject. In some embodiments, the peptide induces, activates, or stimulates the proliferation of Hormad1-specific T cells in the subject. The subject may have cancers such as, for example, 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. Cancers described herein, such as 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, may also be excluded from the methods of this disclosure. Cancer may include cancers that are positive for the expression of the peptide. In some embodiments, the subject is determined to have cells that are positive for the expression or overexpression of the peptide. In some embodiments, the method further includes a step of administering autologous dendritic cells to the subject, wherein the peptide is bound to or presented by the autologous dendritic cells. In some embodiments, the peptide and artificial antigen-presenting cells (aAPCs) are administered to the subject, wherein the peptide is bound to or presented by the aAPCs. In some embodiments, the peptide is functionally linked to the artificial antigen-presenting cells (aAPCs). The term "functionally linked" refers to a situation in which two components are combined or can be combined to form a complex. For example, the components may be covalently linked to and / or on the same polypeptide, such as a fusion protein, or the components may have some degree of binding affinity to each other, such as binding affinity resulting from van der Waals forces. In some embodiments, the subject is human. In some embodiments, the method further includes a step of administering at least a second anti-cancer therapy.The second anti-cancer therapy may be selected from a group consisting of chemotherapy, radiation therapy, immunotherapy, or surgery.

[0014] Another aspect of this disclosure relates to a method for activating or expanding Hormad1-specific T cells, comprising the steps of (a) obtaining a starting population of cells from a mammalian subject, preferably from a blood sample derived from a mammalian subject, wherein the starting population of cells comprises T cells; and (b) contacting the starting population of cells ex vivo with a Hormad1-derived peptide as described herein or above (e.g., SEQ ID NO: 5), thereby activating, stimulating the proliferation of, and / or expanding the proliferation of Hormad1-specific T cells in the starting population. In some embodiments, the contacting step is further defined as co-culturing the starting population of T cells with antigen-presenting cells (APCs), the APCs being able to present the Hormad1-derived peptide on their surface. In some embodiments, the APCs are dendritic cells. In some embodiments, the dendritic cells are autologous dendritic cells obtained from a mammalian subject. In some embodiments, the contacting step is further defined as co-culturing an artificial antigen-presenting cell (aAPC) with a starting population of T cells. In some embodiments, artificial antigen-presenting cells (aAPCs) include or consist of poly(lactide-coglycolide) (PLGA), K562 cells, paramagnetic beads coated with CD3 and CD28 agonist antibodies, beads or microparticles coupled with HLA dimers and anti-CD28, or nano-sized aAPCs (nano-aAPCs) having a diameter of less than 100 nm. In some embodiments, T cells are CD8 + T cells or CD4 +They are T cells. In some embodiments, the T cells are cytotoxic T lymphocytes (CTLs). In some embodiments, the starting population of cells comprises or consists of peripheral blood mononuclear cells (PBMCs). In some embodiments, the method further comprises isolating or purifying T cells from peripheral blood mononuclear cells (PBMCs). In some embodiments, the mammalian subject is human. The method may further comprise re-injecting or administering activated or expanded Hormad1-specific T cells to the subject.

[0015] Yet another aspect of the invention relates to Hormad1-specific T cells activated or expanded by the methods described herein or above.

[0016] Another aspect of the invention relates to a pharmaceutical composition comprising Hormad1-specific T cells activated or expanded by the methods described herein or above.

[0017] Another aspect of this disclosure relates to engineered T cell receptors (TCRs) having antigen specificity for Hormad1 or SEQ ID NO: 5, where the TCRs comprise amino acid sequences of SEQ ID NO: 6, 7, 8, 9, 10, and / or 11. The engineered TCRs may include TCRα CDR3 comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8, and TCRβ CDR3 comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. The manipulated TCR contains an amino acid sequence with sequence identity of 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% with SEQ ID NO:8, TCRα CDR3, and SEQ ID TCRβ CDR3 may include 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 with NO:11. In some embodiments, the TCR may include TCRα CDR1 and / or CDR2, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO:6 and / or 7, and TCRβ CDR1 and / or CDR2, each containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO:9 and / or 10.In some embodiments, the TCRs include TCRα CDR1 and / or CDR2, each containing amino acid sequences 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 with SEQ ID NO: 6 and / or 7 respectively, and SEQ ID NO:9 and / or 10 and TCRβ CDR1 and / or CDR2 containing amino acid sequences 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. In some embodiments, the manipulated TCR includes (i) an α-chain variable region having the amino acid sequence of SEQ ID NO: 13 or 2, or a sequence having at least 90% sequence identity with 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 with SEQ ID NO: 15 or 4. The manipulated TCR may bind to SEQ ID NO: 5 when bound to HLA-A2. The manipulated TCR may bind to the MHC / peptide of SEQ ID NO: 5 bound to HLA-A2. In some embodiments, the TCR includes an α-chain variable region having at least 95% identity with the amino acid sequence of SEQ ID NO: 13 or 2, and / or a β-chain variable region having at least 95% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, the TCR includes an α-chain variable region having at least 99% identity with the amino acid sequence of SEQ ID NO: 13 or 2, and / or a β-chain variable region having at least 95% identity with the amino acid sequence of SEQ ID NO: 15.In some embodiments, the TCR comprises an α-chain variable region having at least 95% identity with the amino acid sequence of SEQ ID NO: 13 or 2, and / or a β-chain having at least 99% identity with the amino acid sequence of SEQ ID NO: 15 or 4. In some embodiments, the TCR comprises an α-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-stranded TCR (scTCR) in which the α-chain and β-chain are covalently linked via a mobile linker. In some embodiments, the TCR comprises or consists of a bispecific TCR. The bispecific TCR may include an scFv that targets or selectively binds to CD3.

[0018] Another aspect of this disclosure relates to polyvalent TCR complexes comprising multiple TCRs as described herein or above. In some embodiments, the polyvalent TCR comprises two, three, four or more TCRs linked together. In some embodiments, the polyvalent TCR is present in a lipid bilayer, in liposomes, or attached to nanoparticles. In some embodiments, the TCRs are linked together via linker molecules or unnatural disulfide bonds.

[0019] A further aspect of the present invention relates to nucleic acids comprising or consisting of nucleotide sequences encoding TCRs as described herein or above. In some embodiments, the nucleic acid comprises cDNA encoding a TCR.

[0020] Another aspect of this disclosure relates to an expression vector comprising the nucleic acid described above. The vector may contain both the TCRα gene and the TCRβ 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).

[0021] Another aspect of the present invention relates to host cells engineered to express a TCR as described herein or above, preferably wherein the host cells comprise an expression vector as described herein or above. In some embodiments, the cells are T cells, NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), or induced pluripotent stem (iPS) cells. In some embodiments, the host cells are immune cells. In some embodiments, the host cells are isolated from the umbilical cord. In some embodiments, the T cells are CD8+ T cells, CD4+ T cells, or γδ T cells. In some embodiments, the T cells are regulatory T cells (Tregs). In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic.

[0022] A further aspect of this disclosure relates to methods for manipulating the host cells described herein or above, including the step of contacting immune cells with nucleic acids or expression vectors described herein or above. In some embodiments, the immune cells are T cells or peripheral blood lymphocytes. In some embodiments, the contacting step is further defined as transfecting or transmuting. Transfecting may include electroporating immune cells with RNA encoding a TCR as described herein or above. The method may further include the step of transmuting immune cells with a viral supernatant prepared from an expression vector as described herein or above. In some embodiments, the immune cells are stimulated lymphocytes (e.g., human lymphocytes). In some embodiments, stimulating includes contacting immune cells with OKT3 and / or IL-2, or incubating immune cells in OKT3 and / or IL-2. In some embodiments, the method further includes the step of sorting the immune cells to isolate TCR-manipulated T cells. The method may further include the step of performing T cell cloning by serial dilution. In some embodiments, the method further includes the expansion and proliferation of T cell clones by a rapid expansion and proliferation protocol.

[0023] Another aspect of this disclosure relates to a method for treating Hormad1-expressing cancer in a mammalian subject, comprising the step of administering an effective amount of TCR-engineered cells as described herein or above to the subject. In some aspects, the TCR-engineered cells are T cells or peripheral blood lymphocytes. In some aspects, the T cells are CD8+ T cells, CD4+ T cells, or Tregs. In some aspects, the cancer is breast cancer, lung cancer, esophageal carcinoma (esophageal 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, head cancer, or cervical cancer. In some aspects, the cancer is a solid tumor. The subject may be human. In some aspects, the TCR-engineered cells are autologous or allogeneic to the subject. The method may further comprise lymphocyte depletion of the subject prior to administration of Hormad1-specific T cells. In some embodiments, lymphocyte depletion includes the administration of cyclophosphamide and / or fludarabine. The method may further include a step of administering a second anticancer therapy. In some embodiments, the second therapy is chemotherapy, immunotherapy, surgery, radiotherapy, or biological therapy. In some embodiments, TCR-manipulated cells and / or at least the second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, locally, or by direct injection or perfusion. In some embodiments, the subject is determined or diagnosed to have cancer cells that overexpress Hormad1.

[0024] In some aspects, methods are provided for the treatment of cancer (e.g., breast cancer, lung cancer, etc.), including a step of immunizing a subject with a purified tumor antigen or an immunodominant tumor antigen-specific peptide, such as Hormad1 peptide (SEQ ID NO: 5). In some embodiments, the peptide can be injected in a solution (e.g., saline solution) as a vaccine or to induce an immune response to the peptide. Adjuvants can be included in the formulation or solution to enhance the solubility of the peptide and / or to increase the immune response in the subject (e.g., Massarelli et al. 2019). Peptide-pulsed mature dendritic cells can be administered to the subject in some embodiments. Approaches that can be used to induce an immune or anti-cancer response to the peptide in the subject include, for example, Wen et al. (2019) and Massarelli et al. (2019). In some embodiments, Hormad1 peptide (SEQ ID NO: 5) is conjugated to or presented by autologous dendritic cells that can be reinjected into the subject or a human patient.

[0025] Throughout this application, the term “about” is used in accordance with its plain and common sense in the field of cell and molecular biology to indicate that the value includes the standard deviation of error with respect to the apparatus or method used to determine that value.

[0026] When used with the term "comprising," the use of the words "a" or "an" can mean "one," but can also mean "one or more," "at least one," and "one or more."

[0027] As used herein, the terms “or” and “and / or” are used to describe multiple components in combination or mutually exclusive. For example, “x, y, and / or z” may 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 particularly intended that x, y, or z may be excluded from the embodiments.

[0028] The words “comprising” (and any form of “comprising,” such as “comprise” and “comprises”), “having” (and any form of “having,” such as “have” and “has”), “including” (and any form of “including,” such as “includes” and “include”), “characterized by” (and any form of “including,” such as “characterized as”), or “containing” (and any form of “containing,” such as “contains” and “contain”) are inclusive or non-restrictive and do not exclude further, unquoted stages of elements or methods.

[0029] The compositions and methods for their use may "comprise," "consist of," or "consist of" any of the components or steps disclosed in this application as a whole. The phrase "consist of" excludes any element, step, or component not specified. The phrase "consist of" limits the scope of the described subject matter to those that do not substantially affect the particular material or step and its basic and novel features. Aspects described in the context of the term "comprising" are also intended to be implemented in the context of the terms "consisting of" or "consisting essentially of."

[0030] It is particularly intended that any limitations discussed in relation to one aspect of the present invention may apply to any other aspect of the present invention. Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to prepare or utilize any composition of the present invention. Aspects of the aspects described in the examples may also be implemented elsewhere in different examples or elsewhere in this application, for example, in the context of aspects discussed in the summary of the invention, the detailed description of aspects, the claims, and the legend of the figures.

[0031] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, while the detailed description and specific examples illustrate certain aspects of the present invention, it should be understood that they are merely illustrative, as various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0032] The patent or application file must include at least one drawing drawn in color. A copy of this patent or patent application publication containing the color drawing will be provided by the authorities upon request and payment of the required fees.

[0033] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings together with the detailed description of the specific embodiments presented herein.

[0034] [Figure 1A] Figures 1A-1D. Hormad1 expression in normal and tumor tissues. (Figure 1A) Hormad1 expression in normal tissue. (Figure 1B) High Hormad1 expression in esophageal cancer, lung cancer, and head and neck cancer. (Figure 1C) High Hormad1 expression in cervical cancer, bladder cancer, and acute myeloid cancer. (Figure 1D) High Hormad1 expression in melanoma and gastric cancer. [Figure 1B] See the explanation in Figure 1A. [Figure 1C] See the explanation in Figure 1A. [Figure 1D] See the explanation in Figure 1A. [Figure 2] T cell receptor (TCR) repertoire analysis of the Hormad1-56 A12 CTL cell line. TCR α and β chains were cloned from Hormad1-56 A12 CTLs using 5'-RACE PCR. Both α and β chains were sequenced and annotated using the IMGT / V-QUEST tool. The CDR3 sequences and TCR usages of the α and β chains are shown. [Figure 3] Generation of Hormad1-56 antigen-specific T cell receptor-modified T cells (TCR-T). Full-length TCR α and β chains were inserted into the retroviral vector pMSGV3, and this recombinant retroviral vector was then used to infect peripheral blood mononuclear cells (PBMCs). An empty retroviral vector was used as a control. After infection, a CD8+ / tetramer+ population was observed by flow cytometry (FCM). After tetramer induction, sorting, and expansion, high-purity TCR-T cells were generated. [Figure 4A]Figures 4A-4F. Hormad1-56 TCR-T cell death assay using different targets. (Figure 4A) Peptide titer assay: T2 cells were pulsed with Hormad1-56 peptide at various concentrations as the target. The effector-to-target (E:T) ratio was 20:1. (Figures 4B-F) Tumor-targeted cell death assay: (Figure 4B) Tumor cell lines H1395 (HLA-A2+, Hormad1+) and H522 (HLA-A2+, Hormad1-), (Figure 4C) Tumor cell lines H1299 (HLA-A2-, Hormad1+) and H1299-A2 (HLA-A2 forced expression, Hormad1+), (Figure 4D) Tumor cell lines H1355 (HLA-A2+, Hormad1+) and H1755 (HLA-A2+, Hormad1-), (Figure 4E) K562-A2 cell line with forced expression of eGFP control gene or Hormad1 gene, or (Figure 4F) H522 tumor cell line with forced expression of eGFP control gene or Hormad1 gene were co-cultured with Hormad1-56 TCR-T cells. In tumor-targeted tumor death assays, the effector-to-target (E:T) ratio ranged from 40:1 to 1.25:1. The lysis capacity of Hormad1-56 TCR-T against different targets was detected by Cr51 release assay (CRA). [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 4D] See the explanation in Figure 4A. [Figure 4E] See the explanation in Figure 4A. [Figure 4F] See the explanation in Figure 4A. [Figure 5-1]Functional detection of Hormad1-56 TCR-T cells by intracellular cytokine staining (ICS) assay. Hormad1-56 TCR-T cells were co-cultured with H522, H1395, H1755, H1355, DFC1032, HSAEC2-KT, H1299, H1299-A2, H522-eGFP, H522-Hormad1, K562-A2-eGFP, and K562-A2-Hormad1 in an E:T ratio of 10:1. After overnight co-culture, the TCR pathway downstream activation markers CD137, CD69, IFN-γ, and TNF-α were detected by ICS assay. When Hormad1-56 TCR-T cells were co-cultured with positive targets H1395, H1355, H1299-A2, H522-Hormad1, and K562-A2-Hormad1, the levels of CD137, CD69, IFN-γ, and TNF-α in Hormad1-56 TCR-T cells were significantly enhanced compared to negative controls. [Figure 5-2] See the explanation in Figure 5-1. [Figure 6A] Figures 6A-6B. Complete sequences of Hormad1-TCR. (Figure 6A) Hormad1 CTL A12 TCR (TRAV4*01 F, TRBV13*01 F) α-chain complete sequence. (SEQ ID NO: 2) (Figure 6B) Hormad1 CTL A12 TCR (TRAV4*01 F, TRBV13*01 F) β-chain complete sequence. (SEQ ID NO: 4) Blue: signal peptide; Yellow: variable region; Red: CDR1, CDR2, CDR3; Black: constant region. [Figure 6B] See the explanation in Figure 6A. [Modes for carrying out the invention]

[0035] Description of exemplary embodiments In several aspects, Hormad1-derived peptides recognized by MHC I (HLA-A2) can be provided and 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. These expanded or activated antigen-specific T cells can be used in cancer treatments such as adoptive cell transfer therapy. Therefore, various cancers expressing Hormad1, such as lung cancer, cervical cancer, esophageal carcinoma, head and neck cancer, leukemia, or solid tumors, can be treated in mammalian subjects (e.g., humans).

[0036] In a further context, cloned T cell receptor (TCR) sequences (e.g., SEQ ID NO: 1-4) capable of binding to the Hormad1-derived peptide / HLA-A2 complex are provided. The TCRs of this disclosure can be used to produce T cells that recognize the Hormad1-derived peptide / HLA-A2 complex. Such T cells include engineered T cells (TCR-T) that express the TCR. These engineered T cells can be used to treat cancer. Related soluble TCRs (sTCRs) and single-stranded TCRs (scTCRs) are also provided and can be used to produce engineered T cells that can be used in adoptive cell transfer therapy to treat cancer.

[0037] The provided peptide and TCR, or the antigen-binding domain or functional fragment of the TCR, can be incorporated into various further constructs. For example, in some embodiments, the antigen-binding domain of the TCR can be incorporated into a chimeric antigen receptor (CAR). Using the peptide (e.g., SEQ ID NO:5), MHC-peptide multimers or tetramers (e.g., HLA-A2 / peptide tetramer) can also be constructed, and the peptide can be incorporated into immunogenic compositions.

[0038] I. Manipulated T cell receptors In various contexts, 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 domain of the TCR (such as CDR1, CDR2, and CDR3 shown in Figures 6A-B) can be included as an extracellular domain containing the antigen-binding domain in soluble TCRs (sTCRs) or chimeric antigen receptors (CARs). In some contexts, the TCRs are isolated or purified TCRs. The polynucleotides encoding the TCRs can be transfected into cells (e.g., autologous or allogeneic cells) that can be used in adoptive cell transfer therapy, also known as "adoptive cell therapy."

[0039] In some embodiments, for example, the T cells of this disclosure (e.g., CD4 + T cells, CD8 +Host cells such as 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 engineered TCRs and / or receptors such as chimeric antigen receptors (CARs). For example, autologous or allogeneic cells (e.g., isolated from umbilical cord or a healthy donor) can be modified to express a T cell receptor (TCR) that has antigen specificity for a short peptide derived from a cancer antigen (e.g., Hormad1 and SEQ ID NO: 5), for example, when presented in association with a specific MHC allele (e.g., HLA-A2). In certain embodiments, the TCR has antigen specificity for the Hormad1-derived peptide (SEQ ID NO: 5) / HLA-A2 complex. In some embodiments, the engineered TCR includes the CDR1, CDR2, and CDR3 regions of the TCRα and TCRβ chains, as shown in Figures 6A-B. In some embodiments, the manipulated TCR has an α chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 2 and / or a β chain containing an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 4. In some embodiments, the TCR has an α chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 1 and / or a β chain having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 3. Suitable methods for modifying amino acid sequences (e.g., for introducing substitutions, deletions, or insertions) are known in the art.

[0040] A. T cell receptor (TCR) In some aspects, recombinant T cell receptors (TCRs) are provided herein. “T cell receptors” or “TCRs” generally comprise variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively) and are capable of specifically binding to an antigenic peptide conjugated to an MHC receptor. In some embodiments, the TCR is in an αβ form and is referred to as TCRαβ. In certain embodiments, the engineered TCR has an α chain variable region at SEQ ID NO: 2 and / or a β chain variable region at SEQ ID NO: 4. In some embodiments, the TCRα chain is encoded by a nucleic acid containing or consisting of SEQ ID NO: 1, and the β chain is encoded by a nucleic acid containing or consisting of SEQ ID NO: 3.

[0041] Aspects of this disclosure relate to engineered T cell receptors. The term "engineered" means a T cell receptor having a TCR variable region grafted onto the TCR constant region for the purpose of creating chimeric polypeptides that bind to the peptides and antigens of this disclosure. In certain embodiments, the TCR is used for cloning, enhancement of expression, detection, or therapeutic control of a construct, but includes intervening sequences not present in the endogenous TCR, such as multicloning sites, linkers, hinge sequences, modified hinge sequences, modified transmembrane sequences, detection polypeptides or molecules, or therapeutic controls that may enable the selection or screening of cells containing the TCR.

[0042] In some embodiments, the TCR includes non-TCR sequences. Therefore, certain embodiments relate to TCRs having sequences not derived from TCR genes. In some embodiments, the TCR is a chimera in that it includes sequences typically found in TCR genes, but also includes sequences from at least two TCR genes that are not necessarily found together in nature.

[0043] The TCRs provided below are identified herein as selectively binding to Hormad1-derived peptides (e.g., SEQ ID NO: 5) / HLA-A2 complexes. α-chain DNA sequence (SEQ ID NO: 1) TIFF2026053605000002.tif106160α-chain protein sequence (SEQ ID NO: 2): TIFF2026053605000003.tif33159β-chain DNA sequence (SEQ ID NO: 3): TIFF2026053605000004.tif128159β-chain protein sequence (SEQ ID NO: 4): TIFF2026053605000005.tif40159 HLA-A2-restricted peptide derived from Hormad1 (SEQ ID NO: 5): YLDDLCVKI α-chain CDR1 peptide (SEQ ID NO: 6): NIATNDY α-chain CDR2 peptide (SEQ ID NO: 7): GYKTK α-chain CDR3 peptide (SEQ ID NO: 8): LVGARGTALIF β-chain CDR1 peptide (SEQ ID NO: 9): PRHDT β-chain CDR2 peptide (SEQ ID NO: 10): FYEKMQ β-chain CDR3 peptide (SEQ ID NO: 11): ASSPTGQGSYEQY α-chain variable region DNA sequence (SEQ ID NO: 12): TIFF2026053605000006.tif47159α-chain variable region protein sequence (SEQ ID NO: 13): TIFF2026053605000007.tif11157β-chain variable region DNA sequence (SEQ ID NO: 14): TIFF2026053605000008.tif47159β-chain variable region protein sequence (SEQ ID NO: 15): TIFF2026053605000009.tif11158

[0044] Unless otherwise specified, the term “TCR” should be understood to encompass both full-length native TCR polypeptides and their functional fragments in various combinations, including αβ or γδ forms. As used herein, a “functional” TCR or fragment can form a full-length or truncated TCR that retains the ability to bind its homologous peptide, presented in association with a suitable MHC allele (e.g., α with β, or γ with δ), by binding with its homologous subunit (e.g., α with β, or γ with δ).

[0045] Therefore, for the purposes of this specification, references to TCRs include any TCR or TCR fragment capable of binding to an antigen peptide, such as the antigen-binding portion of a TCR that binds to a specific antigen peptide (i.e., an MHC-peptide complex) bound in an MHC molecule. The terms “antigen-binding portion” or “antigen-binding fragment” of a TCR are used interchangeably herein to refer to a molecule containing a portion of a TCR that binds to an antigen (e.g., an MHC-peptide complex) to which a full-length TCR binds.

[0046] The variable domain of the TCR chain is generally understood to form a loop or complementarity-determining region (CDR) similar to those present in immunoglobulins that confer antigen recognition; in TCRs, the CDR determines peptide specificity by forming the binding site of the TCR molecule. Typically, as with immunoglobulins, the CDR is separated by a framework region (FR) (see, e.g., Jores et al., 1990; Chothia et al., 1988; also see Lefranc et al., 2003). The CDR3 regions on the α and β chains of the TCR are generally understood to be involved in the binding of the processed antigen peptide. In some embodiments, the variable region of the β chain may include an additional hypervariability (HV4) region.

[0047] Although α / β and γ / δ TCRs are structurally similar, the T cells expressing them may have different anatomical locations or functions. As will be understood by those skilled in the art, TCRs are found on the surface of T cells (or T lymphocytes) capable of recognizing antigen-derived peptides bound to major histocompatibility complex (MHC) molecules. TCRs contain distinct regions including a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (e.g., Janeway et al, Immunobiology: The Immune System in Health and Disease, 3). rd (See Ed., Current Biology Publications, p. 433, 1997). The TCR α and β chains can associate with the invariant proteins of the CD3 complex, which are involved in signal transduction.

[0048] In some embodiments, the TCR comprises a functional fragment of the Hormad1-TCR. In some embodiments, the functional fragment comprises a constant domain and a variable domain of the Hormad1-TCR. Similar to immunoglobulins, the extracellular portion of the TCR chain (e.g., α chain, β chain) consists of two immunoglobulin domains, i.e., the N-terminal variable domain (e.g., V a Typically, Kabat numbering is used. 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 Based on the ed., amino acid numbers 1-116, and one constant domain adjacent to the cell membrane (e.g., α-chain constant domain or C aThe TCR domain may include, typically based on Kabat, amino acid numbers 117–259, and the β-chain constant domain (typically based on Kabat, amino acid numbers 117–295). For example, in some cases, the extracellular portion of the TCR formed by two chains (e.g., either αβ or γδ form) may include two membrane-proximal constant domains and two CDR-containing membrane-distal variable domains. The constant domains of the TCR domain include short ligation sequences in which cysteine ​​residues form disulfide bonds, resulting in a linkage between the two chains. In some embodiments, it may be possible to improve TCR gene transfer by adding a single cysteine ​​to each receptor chain to promote the formation of further interchain disulfide bonds, as described, for example, in Cohen et al. (2007).

[0049] A CDR may also contain 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 consecutive amino acid residues (or any range derivable therein) adjacent to one or both sides of a particular CDR sequence in the context of the variable region of a TCR-a or TCR-b polypeptide; therefore, one or more additional amino acids may be present at the N-terminus or C-terminus of a particular CDR sequence, such as those shown in the variable regions of SEQ ID NO: 13 and 15. Alternatively, or in combination, a CDR may be a fragment of a CDR described herein, which may lack at least 1, 2, 3, 4, or 5 amino acids from the C-terminus or N-terminus of a particular CDR sequence.

[0050] In some embodiments, each TCR chain contains a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the TCR can associate with other molecules such as CD3. For example, a TCR containing both a constant domain and a transmembrane domain can enable the protein to be immobilized in the cell membrane and to associate with the invariant subunit of the CD3 signaling machinery or complex.

[0051] CD3 is a multi-subunit complex containing distinct chains: γ, δ, ε, and ζ. For example, in mammals, this complex may contain one CD3γ chain, one CD3δ chain, two CD3ε chains, and a homodimer of CD3ζ chains. CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily. The transmembrane domains of CD3γ, CD3δ, and CD3ε chains are negatively charged, a property that allows them to associate with positively charged T cell receptor chains. The intracellular tails of CD3γ, CD3δ, CD3ε, and CD3ζ chains each contain conserved motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs are conserved amino acid sequences that can be repeated and are involved in the signaling ability or signal transduction of the TCR complex. These auxiliary molecules possess negatively charged transmembrane domains and play a role in transmitting signals from the TCR to the cell. The CD3 chain and ζ chain, together with the TCR, form what is known as the T cell receptor complex (TCR complex).

[0052] In some embodiments, TCR comprises a heterodimer containing one TCRα polypeptide and one TCRβ polypeptide. TCR may also comprise a heterodimer containing one TCRγ polypeptide and one TCRδ polypeptide. In some embodiments, TCR comprises a single-stranded TCR (scTCR). In some embodiments, the polypeptides of the TCR heterodimer are covalently bonded. In some embodiments, the covalent bond is due to one or more disulfide bonds. In some embodiments, one or more disulfide bonds include naturally occurring disulfide bonds, such as those found in natural TCR. In some embodiments, one or more disulfide bonds include disulfide bonds that are not naturally occurring and are not found in natural TCR.

[0053] The TCRs of this disclosure can be expressed in cells such as T cells by transfecting cells with nucleic acids encoding the TCRs using various methods, as will be understood 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, including electrotransfection (e.g., Zhang et al., 2018), are used to transfect T cells (as described, for example, Riet et al., 2013).

[0054] B. Soluble TCR In some embodiments, this disclosure provides soluble TCRs that may include variable regions of TCRs specific to Hormad1-derived peptides provided herein (e.g., SEQ ID NO: 13 and 15). Soluble TCRs are potentially useful not only for investigating specific TCR-MHC interactions but also as diagnostic tools for detecting infections or autoimmune disease biomarkers. Soluble TCRs also have applications in staining, for example, to stain cells for the presence of specific peptide antigens presented in association with MHC. Similarly, soluble TCRs can be used to deliver therapeutic agents, such as cytotoxic or immunostimulatory compounds, to cells presenting specific antigens. Soluble TCRs may also be used to inhibit T cells, for example, T cells that react to autoimmune peptide antigens.

[0055] In the context of this application, "solubility" is defined as the ability of TCR to be purified as a monodisperse heterodimer at a concentration of 1 mg / ml in phosphate-buffered saline (PBS) (KCl 2.7 mM, KH2PO4 1.5 mM, NaCl 137 mM, and Na2PO4 8 mM, pH 7.1-7.5. Life Technologies, Gibco BRL), with more than 90% of the TCR remaining as a monodisperse heterodimer after incubation at 25°C for 1 hour.

[0056] In some aspects, the present disclosure provides a soluble T cell receptor (sTCR) comprising (i) all or part of a TCRα chain (e.g., SEQ ID NO: 1 or 2), excluding its transmembrane domain, and (ii) all or part of a TCRβ chain (e.g., SEQ ID NO: 3 or 4), excluding its transmembrane domain, wherein (i) and (ii) each comprise at least a part of the functional variable and constant domains of the TCR chain and are linked by a disulfide bond between constant domain residues not present in the native TCR. In some aspects, the soluble TCR comprises a TCRα or γ chain extracellular domain dimerized to a TCRβ or δ chain extracellular domain by a pair of C-terminal dimerization peptides such as leucine zippers (International Patent Publication No. WO 99 / 60120; U.S. Patent No. 7,666,604).

[0057] In some embodiments, the entire antigen-binding region comprising the variable region of the TCR (see, e.g., FIGS. 6A - B) can be included in the sTCR. The sTCR can be a single-chain T cell receptor (scTCR), where the variable regions (Vα and Vβ) from the α and β chains are covalently linked via a flexible linker, and the end of the variable region (typically the end of Vβ not linked to the linker) is covalently linked to a therapeutic compound (e.g., a toxin, a chemotherapeutic agent, etc.) or a contrast agent. The sTCR can recognize intracellular or extracellular epitopes when presented by MHC, and the sTCR can be used for the identification of native peptide ligands in diseases (see, e.g., Walseng et al., 2015; Boulter et al., 2005). Thus, the sTCR can be administered to a subject such as a human patient to visualize tumor cells or deliver a therapeutic compound to cancer cells to treat cancer. 131Various therapeutic molecules or toxins, such as Hormad1-derived peptides / HLA-A2 complexes, auristatin, maytansine, calicheamicin, STING agonists, cytokines, chemokines, costimulatory agonists (e.g., OX40), or other chemotherapeutic agents, can be delivered by sTCRs to cells such as cancer cells expressing Hormad1-derived peptides / HLA-A2 complexes. In this way, sTCRs can be used for targeted delivery of therapeutic molecules to tumor sites. In some embodiments, sTCRs contain or are covalently bound to fluorescent or radioactive probes.

[0058] The soluble TCRs of this disclosure, which may be of human origin or produced in human cells, may be provided in substantially pure form or as purified or isolated preparations. For example, they may be provided in a form that is substantially free of other proteins.

[0059] The multiple soluble TCRs of this disclosure may be provided as multivalent complexes. Therefore, in one aspect, this disclosure provides a multivalent T cell receptor (TCR) complex comprising multiple soluble T cell receptors as described herein. Each of the multiple soluble TCRs is preferably identical. The multivalent TCR may comprise two or more ligand-bound TCRα / β subunits (see, e.g., Schamel et al., 2005).

[0060] A polyvalent TCR complex generally comprises a multimer of two, three, four, or more T cell receptor molecules linked to one another (e.g., covalently or otherwise) via a linker molecule. Suitable linker molecules include, but are not limited to, polyvalent adhesion molecules such as avidin, streptavidin, neutraavidin, and exlaavidin, each having four binding sites to biotin. Thus, biotinylated TCR molecules can be used to form a multimer of T cell receptors 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 construct the multimer, and on the presence or absence of any other biotinylated molecules. Preferred multimers are dimeric, trimer, or tetrameric TCR complexes.

[0061] TCRs or polyvalent TCR complexes can be attached to solid structures, which are membrane structures (e.g., liposomes) or preferably particles such as beads (e.g., latex beads). In some embodiments, the structure is coated with T cell receptor multimers rather than individual T cell receptor molecules. In the case of liposomes, the T cell receptor molecules or their multimers can be attached to the membrane or otherwise associated with the membrane. Techniques for this are well known to those skilled in the art.

[0062] A label or other part, such as a toxic or therapeutic part, may be included in a polyvalent TCR complex. For example, the label or other part may be included in a mixed molecular polymer. An example of such a polymeric molecule is a tetramer containing three TCR molecules and one peroxidase molecule. This can be achieved by mixing the TCRs and enzymes in a molar ratio of approximately 3:1 to produce a tetrameric complex and then isolating the desired complex from a complex that does not contain molecules in the correct proportions. These mixed molecules may contain any combination of molecules, provided that steric hindrance does not impair, or significantly impair, the desired function of the molecules. The arrangement of binding sites on the streptavidin molecule may be suitable for a mixed tetramer because the likelihood of steric hindrance is low.

[0063] In some embodiments, the peptides provided herein (e.g., SEQ ID NO: 5) can be used to construct 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 after pulsing a professional APC with a specific Hormad1 peptide, Hormad1 protein, or a nucleotide sequence encoding a specific Hormad1 peptide or Hormad1 protein in vitro. In some cases, MHC-peptide tetramers can be used to visualize T cells in tissues (e.g., Dileepan et al., 2015). MHC multimer induction methods can also be used to facilitate the isolation of functional T cell receptors from single cells that may be used in immunotherapy. For example, direct isolation of paired full-length TCR sequences from non-extending antigen-specific T cells can be achieved using PCR-based T cell receptor single-cell analysis (TCR-SCAN) (e.g., Dossinger et al., 2013). Therefore, using a multimer-inducible sorting strategy, T cells that selectively identify the Hormad1 peptide (e.g., SEQ ID NO:5) can be isolated from PBMCs of HLA-A2-positive patients or from T cells stimulated (e.g., with the peptide or aAPC). After injection, antigen-specific T cells can be tracked in tetramers or multimers for evaluation of long-term persistence in vivo.

[0064] The TCRs (or their multivalent complexes) of this disclosure may be alternatively or additionally linked to therapeutic agents, such as toxic moieties for use in cell death, or immunostimulants such as interleukins or cytokines (e.g., covalently or otherwise). The multivalent TCR complexes of this disclosure may have enhanced binding ability to TCR ligands compared to non-multivalent T cell receptor heterodimers. Thus, in some embodiments, the multivalent TCR complexes may be used to track or target cells presenting specific antigens in vitro or in vivo. Therefore, the TCRs or multivalent TCR complexes may be provided in formulations pharmaceutically acceptable for in vivo use.

[0065] This disclosure also provides a method for delivering a therapeutic agent to target cells, the method comprising the step of bringing potential target cells into contact with a TCR or a multivalent TCR complex under conditions that allow the TCR or multivalent TCR complex to adhere to the target cells, the TCR or multivalent TCR complex being specific to a TCR ligand and to which the therapeutic agent is bound.

[0066] In some embodiments, soluble TCRs or multivalent TCR complexes can be used to deliver therapeutic agents to the location of cells presenting a specific antigen. This may be useful, for example, for the treatment of tumors. The therapeutic agent may be delivered to exert its effect not only on the cells to which it binds, but also locally (for example, chemotherapeutic agents, radioactive materials, or enzyme agents may produce a local effect near or on the tumor). Thus, one particular strategy envisions an antitumor molecule linked to a T cell receptor or multivalent TCR complex specific to the tumor antigen.

[0067] Many therapeutic agents, such as radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin), can be used for this application. To reduce or limit toxic effects at the desired site, the toxin may be delivered in liposomes ligated to streptavidin so that the compound is released slowly. This may help reduce the damaging effects during transport in the body and limit the toxic effects until after the binding of the TCR to the relevant antigen-presenting cells or cells expressing the Hormad1 antigen (e.g., cancer cells).

[0068] Other suitable therapeutic agents include (1) small molecule cytotoxic agents, i.e., compounds having the ability to kill mammalian cells with a molecular weight of less than 700 daltons. Such compounds may also include toxic metals that can have cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that disintegrate or are converted under physiological conditions to release cytotoxic agents. Examples of such agents include cisplatin, meitansine derivatives, rakelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimethrexate glucuronide, auristatin E vincristine, and doxorubicin; and (2) peptide cytotoxic agents, i.e., proteins or fragments thereof that have the ability to kill mammalian cells. Examples include lysine, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNAase and RNAase; (3) Radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more α or β particles or γ rays. An example is iodine-131 ( 131 I) Rhenium-186 ( 186 Re), Indium-111 ( 111 In), Yttrium 90 ( 90 Yt), bismuth 210 and 213 ( 210 Bi and 213Bi), Actinium-225 ( 225 Ac), as well as astatine 213 ( 213 (4) Prodrugs such as antibody-directed enzyme prodrugs; and (5) Immunostimulants, i.e., moieties that stimulate the immune response. Examples include cytokines such as IL-2, chemokines such as IL-8, platelet factor 4, melanoma growth-stimulating proteins, antibodies or fragments such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides.

[0069] The soluble TCRs of this disclosure may 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 (e.g., type 1 diabetes) may be treated using this approach. Knowledge of specific peptide epitopes presented by relevant pMHCs is required for this application.

[0070] The soluble TCRs and / or polyvalent TCR complexes of this disclosure may be used in the preparation of compositions for the treatment of cancer or autoimmune diseases.

[0071] Also provided are methods for treating cancer (e.g., leukemia, lung cancer, esophageal cancer, head and neck cancer, or cervical cancer) or other cancers expressing Hormad1 as described herein) or autoimmune diseases, comprising administering an effective amount of the soluble TCR and / or polyvalent TCR complex of the present invention to a patient in need thereof.

[0072] As is common in anti-cancer and autoimmune therapies, the TCR of this disclosure may be used in combination with other agents for the treatment of cancer or autoimmune disease, and one or more additional therapeutic substances or treatments may be administered to treat other related conditions observed in the patient population.

[0073] C. Bispecific TCR In some embodiments, the TCRs of this disclosure are included in bispecific T cell receptors (TCRs). Bispecific TCRs generally include TCRs that are fused, ligated, or covalently bound to either an scFv or an antibody (e.g., McCromack et al., 2013). In some embodiments, the bispecific TCRs of this disclosure include a Hormad1-targeted TCR and a T cell mobilization antibody domain or scFv (e.g., an scFv to CD3 or other immunomodulatory T cell surface proteins). Bispecific TCRs may enable T cells to be activated and attack tumors regardless of the intrinsic specificity of T cells. A bispecific platform usable with the TCRs of this disclosure includes the TCER® molecule (Immatics, Houston, Texas). A further example of a bispecific TCR is ImmTAC (e.g., Oates et al., 2013).

[0074] D. Chimeric antigen receptor Chimeric antigen receptors (CARs) are engineered receptors that can be expressed by T cells and can bind to antigens such as antigens on cancer cells. CARs generally contain different domains, including an antigen-binding domain, a transmembrane domain, and an endodomain. The endodomain transmits activation and co-stimulatory signals to T cells upon antigen recognition. Chimeric antigen receptor molecules are not naturally occurring and are distinguished by their ability to bind to antigens and to transmit activation signals via immune receptor activation motifs (ITAMs) present in their cytoplasmic endodomain. CAR T cells are T cells that have been genetically modified to express CARs.

[0075] Soluble TCR constructs can be fused to the CAR signaling tail (i.e., the transmembrane domain and endodomain) to instruct T cells to recognize an antigen, as described, for example, by Walseng et al. (2017). Such CAR constructs are referred to as "TCR-CARs." Thus, the CAR may comprise the TCR-binding region of this disclosure (e.g., shown in Figures 6A-B) or a soluble TCR, either covalently bound to the transmembrane domain and endodomain or expressed as a fusion protein with the transmembrane domain and endodomain. The endodomain may comprise, for example, the CD3ζ, CD28 intracellular signaling domain, 4-1BB (CD137), (CD3ζ and CD28), CD27, OX-40 (CD134), DAP10, or 4-1BB.

[0076] II. Adoptive Cell Transfer Therapy Methods for treating or delaying cancer progression in an individual are provided herein, comprising the step of administering an effective dose of antigen-specific immune cells or stem cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, α-β T cells or γ-δ T cells), NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), or induced pluripotent stem (iPS) cells) therapy to the individual, such as Hormad1-specific cell therapy. Adoptive T cell therapy with genetically modified TCR-generated T cells (e.g., expressing TCRs containing one or more SEQ ID NO: 1-4, such as SEQ ID NO: 2 and SEQ ID NO: 4) is also provided herein. In some embodiments, adoptive cell transfer therapy is provided to a subject (e.g., a human patient) in combination with a second therapy, such as chemotherapy, radiotherapy, surgery, or a second immunotherapy.

[0077] The peptide provided herein (e.g., SEQ ID NO: 5) can also be used to create antigen-specific cytotoxic T cell (CTL) cell lines or clones that can be used in adoptive immunotherapy. 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 then co-cultured with T cells for several rounds of stimulation to create antigen-specific CTL cell lines or clones (e.g., Neal et al., 2017). T cells may be expanded ex vivo using various antigen-presenting cells (APCs), and various strategies can be used for antigen loading of dendritic cells to enhance the antitumor response (see, for example, Strome et al., 2002). The resulting autologous CTL cell lines or clones can be used in adoptive cell transfer immunotherapy for the treatment of cancer patients.

[0078] Aspects of this disclosure include methods for obtaining autologous T cells from a subject, methods for generating TCR-engineered immune cells or stem cells, and methods for administering TCR-engineered cells to a subject as immunotherapy targeting cancer cells. In particular, 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, α-β T cells, or γ-δ 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). Several basic approaches for inducing, activating, and expanding functional antitumor effector cells have been described over the past 20 years. These include autologous cells such as tumor-infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells isolated by capturing target cell membranes; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells that have been genetically reprogrammed or "redirected" to express tumor-reactive TCRs or chimeric TCR molecules that exhibit antibody-like tumor recognition ability known as "T bodies" (e.g., Eshhar et al., 1995). These approaches have given rise to numerous protocols for the preparation and immunization of T cells that can be used in the manner described herein.

[0079] A. T-cell preparation and administration In some embodiments, the engineered T cells are autologous (i.e., isolated from the patient being treated). In some embodiments, the engineered T cells are allogeneic. In some embodiments, allogeneic T cells include T cells pooled from multiple donors.

[0080] In some embodiments, T cells originate from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. Most preferably, T cells are human cells. In some embodiments, T cells obtained from umbilical cord blood may have improved antitumor properties compared to T cells obtained from adult donors (e.g., Hiwarkar et al., 2015). The cells are typically primary cells, such as those isolated directly from the subject and / or those isolated and frozen from the subject. In some embodiments, the cells may be one or more subsets of T cells or other cell types, e.g., whole blood-derived T cells, CD4 + cells, CD8 + This includes cells and their subpopulations, defined, for example, by function, activation state, maturity, differentiation, proliferation, recirculation, localization potential and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With respect to the subject being treated, the cells may be allogeneic and / or autologous. In some aspects, for example in the case of ready-made techniques, the cells are pluripotent and / or multipotent, and are stem cells such as induced pluripotent stem (iPS) cells; for example, stem cells or iPS cells can be differentiated into various T cell populations. In some embodiments, the method includes, as described herein, the steps of isolating cells from a subject, preparing them, processing them, culturing them, and / or manipulating them, and reintroducing them to the same patient (if they are autologous) or a different patient (if they are allogeneic) before or after cryopreservation.

[0081] T cells (e.g., CD4 + and / or CD8 + Among the subtypes and subpopulations of T cells, there are naive T cells (T N ) cells, effector T cells (T EFF ), memory T cells (T MEM ) and their subtypes, for example, stem cell memory T (TSC M ), central memory T (TC M), effector memory T (T EM ), or terminal effector memory T cells (T EMRA ), tumor-infiltrating lymphocyte-derived T cells (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, spontaneous and adaptive regulatory T (Treg) cells, helper T cells such as 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.

[0082] In some embodiments, subpopulations of T cells can be created 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 absent or expressed at relatively low levels in certain populations of T cells (e.g., non-memory cells), but present or expressed at relatively high levels in certain other populations of T cells (e.g., memory cells).

[0083] In some aspects, T cells are isolated from PBMC samples by negative selection of markers expressed on non-T cells such as B cells, monocytes, or other leukocytes, such as CD14. In some aspects, CD4 + or CD8 + The selection stage is CD4 + Helper and CD8 + It is used to isolate cytotoxic T cells. Such CD4 + and CD8 + The population can be further subdivided into subpopulations by positive or negative selection of one or more markers expressed or relatively highly expressed in naive, memory, and / or effector T cell subpopulations. Various methods can be used for cell isolation based on marker expression, including magnetically activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS).

[0084] In some embodiments, CD8 +T cells are further enriched or depleted of naive, central memory, effector memory, and / or central memory stem cells by positive or negative selection based, for example, on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM Cell enrichment is performed to enhance efficacy, such as improving long-term survival, expansion, and / or engraftment after administration (see, e.g., Terakura et al., 2012; Wang et al., 2012).

[0085] 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 medium is prepared from the biological sample obtained from the patient (e.g., from a tumor). The single-cell suspension can be obtained by any suitable method, for example, mechanically (e.g., by dissociating the tumor using, for example, gentleMACS® Dissociator, Miltenyi Biotec, Auburn, Calif.) 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 densely packed (e.g., about 2 × 10⁻⁶). 6 Individual lymphocytes are cultured for, for example, about 5 to about 21 days, preferably about 10 to about 14 days. For example, cells may be cultured for 5 days, 5 to 6 days, or 5 to 21 days, or 10 to 14 days.

[0086] In some embodiments, the naked DNA or suitable vector encoding the TCR or CAR of this disclosure can be introduced into target T cells (e.g., T cells obtained from human patients with cancer or other diseases). Methods for stably transfecting T cells by electroporation using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to the DNA encoding the chimeric receptor of the present invention contained in a plasmid expression vector in an appropriate orientation for expression (e.g., Zhang et al., 2018). In some embodiments, the use of naked DNA can reduce the time required to produce T cells expressing the TCR produced via the method of the present invention. Transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009 can be used. Electroporation of RNA encoding the full-length TCRα and β (or γ and δ) strands can be used as an alternative to overcome the long-term problem of self-reactivity caused by the pairing of retrovirally transduced TCR strands with endogenous TCR strands. In some embodiments, nonviral RNA transfection can be used to transiently modify T cells, for example, as described by Riet et al. (Methods Mol Biol. 2013;969:187-201).

[0087] Alternatively, TCRs or chimeric constructs can be introduced into T cells using viral vectors (e.g., retroviral vectors, adenovirus vectors, adeno-associated virus vectors, or lentiviral vectors). Generally, vectors encoding TCRs or CARs used to transfect T cells from a target should not replicate in the target T cells. Numerous virus-based vectors are known, and the number of viral copies maintained within the cell is low enough to maintain the cell's viability. Exemplary vectors include pFB-neo vectors (STRATAGENE®) and vectors based on HIV, SV40, EBV, HSV, or BPV.

[0088] In some embodiments, the TCR nucleotide sequences encoding the α and β chains of this disclosure (e.g., DNA or RNA sequences) (see, e.g., Figures 6A-B; SEQ ID NO 1-4) can be cloned into other expression vectors or plasmids such as retroviruses, lentiviruses, or MSCVs (mouse stem cell viruses) (e.g., adeno-associated virus plasmids). T cells can be genetically modified 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.

[0089] Once it is established that transfected or transduced T cells can express TCR or CAR as surface membrane proteins and at desired levels, it can be determined whether the TCR or chimeric receptor functions in host cells to provide desired signal induction. The transduced T cells can then be reintroduced or administered to a target to activate, implement, and / or induce an antitumor response in the target. To facilitate administration, the transduced T cells may be prepared as a pharmaceutical composition or implant suitable for in vivo administration using a pharmaceutically acceptable carrier or diluent. Means for preparing such compositions or implants are described in the Art (e.g., Remington: The Science and Practice of Pharmacy, 22 nd (See edition, Pharmaceutical Press, 2012). Where appropriate, transduced T cells expressing a TCR or CAR can be formulated in semi-solid or liquid forms, such as capsules, solutions, or injections, in a manner conventional for each route of administration. By means known in the art, the release and absorption of the composition can be inhibited or minimized until the composition reaches the target tissue or organ, or sustained release of the composition can be ensured. Generally, pharmaceutically acceptable forms that do not significantly adversely affect cells expressing a TCR or chimeric receptor are preferred. In some embodiments, transduced T cells can be in a pharmaceutically acceptable composition containing an equilibrium salt solution, such as Hanks' equilibrium salt solution, or ordinary physiological saline.

[0090] Cultured T cells can be pooled and rapidly expanded. Rapid expansion provides at least a 50-fold increase in the number of antigen-specific T cells (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more) over a period of about 10 to 14 days. More preferably, rapid expansion provides at least a 200-fold increase (e.g., 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold or more) over a period of about 10 to 14 days. In some embodiments, allogeneic T cells can be pooled from several donors.

[0091] Expansion and proliferation can be achieved by various methods known in the art. For example, T cells can be rapidly expanded and proliferated using nonspecific TCR stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. Nonspecific TCR stimulation can include OKT3, i.e., mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, NJ), at around 30 ng / ml. Alternatively, T cells can be rapidly expanded and proliferated by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more cancer antigens (including their antigenic portion, such as an epitope, or cells), which may optionally be expressed from a vector in the presence of a T cell growth factor such as 300 IU / ml IL-2 or IL-15, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, with IL-2 being preferred. In vitro-induced T cells rapidly expand and proliferate upon restimulation with the same cancer antigen pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, T cells can be restimulated, for example, with irradiated autologous lymphocytes, or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0092] Autologous T cells can be modified to express T cell growth factors that promote the growth and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable modification methods are known in the art, for example, Sambrook et al., 2001; and Ausubel et al., 1994. In some embodiments, modified autologous T cells express T cell growth factors at high levels. T cell growth factor coding sequences, such as those for IL-12, as well as promoters that can be used to promote high-level expression, are readily available in the art.

[0093] In certain embodiments, T cell growth factors that promote the growth and activation of autologous or allogeneic T cells are administered to the subject simultaneously with or after the autologous T cells. The T cell growth factor can be any suitable growth factor that promotes the growth and activation of autologous T cells. Examples of suitable T cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, for example, 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 IL-2. IL-12 is a preferred T cell growth factor.

[0094] T cells can be administered intravenously, intramuscularly, subcutaneously, percutaneously, intraperitoneally, intrathecally, parenterally, intrathecally, intracavitally, intravenously, intraarterially, via cerebrospinal fluid, or by any implantable or semi-implantable, permanent or degradable device. The appropriate dosage of T cell therapy may be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0095] Intratumoral injection or injection into the tumor vascular system is particularly intended for accessible, isolated solid tumors. Local, site-specific, or systemic administration may also be appropriate. For tumors larger than 4 cm, a volume of approximately 4–10 ml (especially 10 ml) may be administered, while for tumors smaller than 4 cm, a volume of approximately 1–3 ml (e.g., 3 ml) may be used. Multiple infusions delivered as single doses may contain a volume of approximately 0.1–0.5 ml.

[0096] B. Antigen-presenting cells Antigen-presenting cells (APCs) are a group of heterologous 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, break them down into peptides, and present them in combination with major histocompatibility complex (MHC) molecules on their cell surface that can interact with appropriate T cell receptors. APCs are distinguished by their expression of specific MHC molecules. MHC is a large gene complex with multiple loci. MHC loci encode two major classes of MHC membrane molecules, known as class I MHC and class II MHC. T helper lymphocytes generally recognize antigens associated with MHC class II molecules, while T cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. In humans, MHC is called the HLA complex, and in mice, it is called the H-2 complex.

[0097] In some embodiments, the peptide (e.g., SEQ ID NO:5) is recognized by HLA-A2 and can be used to expand and proliferate antigen-specific T cells in vitro. The peptide, or the nucleic acid encoding the peptide, can be used to stimulate antigen-presenting cells (APCs) to induce 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 then co-cultured with T cells for several rounds of stimulation to produce antigen-specific CTL cell lines or clones. Thus, a population of expanding T cells that selectively recognizes the Hormad1-derived peptide / HLA-A2 complex can be adopted and transferred to a patient to treat cancer or induce tumor regression.

[0098] In some cases, artificial antigen-presenting cells (aAPCs) are useful for preparing therapeutic compositions and cell therapy products based on TCRs or CARs. For general guidelines on the preparation and use of antigen-presenting systems, see, for example, U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Patent Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. WO2007 / 103009).

[0099] aAPCs can be used to expand and proliferate T cells expressing TCRs or CARs. During encounter with tumor antigens, signals delivered to T cells by antigen-presenting cells can influence T cell programming and subsequent therapeutic efficacy. This has stimulated efforts to develop artificial antigen-presenting cells that allow for optimal control of the signals delivered to T cells (Turtle et al., 2010). In addition to the antibody or antigen of interest, an aAPC system may include at least one exogenous co-molecule. Any appropriate number and combination of co-molecules may be utilized. Co-molecules may be costimulatory or adhesion molecules. Exemplary costimulatory molecules include CD70 and B7.1 (also known as B7 or CD80), which bind to CD28 and / or CTLA-4 molecules on the surface of T cells, thereby promoting, for example, T cell expansion and proliferation, Th1 differentiation, short-term T cell survival, and cytokine secretion such as interleukin (IL)-2 (see Kim et al., 2004). Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecules (ICAMs) that facilitate cell-to-cell or cell-to-substrate contact. Exemplary adhesion molecules include LFA-3 and ICAM, e.g., ICAM-1. Techniques, methods, and reagents useful for the selection, cloning, preparation, and expression of exemplary accessory molecules, including co-stimulatory and adhesion molecules, are exemplified, for example, in U.S. Patents 6,225,042, 6,355,479, and 6,362,001.

[0100] C. Nucleic acid In one aspect, this disclosure provides nucleic acids encoding isolated TCRs (e.g., sTCRs), CARs, or peptides disclosed herein. For example, nucleic acids may encode polypeptides comprising TCR variable regions having approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the TCR variable regions disclosed herein (e.g., SEQ ID NO: 1-4), or TCR variable regions having 1, 2, 3, or 4 point mutations (e.g., substitution mutations) compared to any one of SEQ ID NO: 1-4. The term “nucleic acid” is intended to include DNA and RNA and may be either double-stranded or single-stranded.

[0101] Therefore, nucleic acids encoding TCRs (e.g., sTCRs), CARs, or peptides may be functionally ligated to a promoter and / or included in an expression vector. TCRs, CARs, or peptides can be produced in a suitable expression system using methods well known in the field of molecular biology. Nucleic acids encoding tumor antigen-specific peptides disclosed herein may be incorporated into any expression vector that ensures good expression of the peptide in a desired environment (e.g., in human immune cells). Possible vectors that can be used include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), as long as the vector is suitable for transforming host cells.

[0102] A recombinant expression vector “suitable for host cell transformation” means that the expression vector comprises a nucleic acid molecule of the present disclosure and a regulatory sequence selected based on the host cell used for expression, wherein such regulatory sequence is functionally linked to the nucleic acid molecule. The terms “operatively linked” and “operably linked” are used interchangeably and are intended to mean that the nucleic acid is linked to the regulatory sequence in such a way that the expression of the nucleic acid is controlled by the regulatory sequence.

[0103] Accordingly, the present invention provides a recombinant expression vector comprising a nucleic acid encoding a TCR, CAR, or soluble peptide that selectively binds to Hormad1, and regulatory sequences necessary for the transcription and translation of the inserted protein sequence. Suitable regulatory sequences may be derived from various sources, including bacterial, fungal, or viral genes (see, for example, the regulatory sequences described in Goeddel, 1990).

[0104] The selection of appropriate regulatory sequences generally depends on the selected host cell and can be easily performed by those skilled in the art. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences, e.g., translation initiation signals. Furthermore, depending on the selected host cell and the vector used, other sequences such as origins of replication, additional DNA restriction sites, enhancers, and sequences that confer transcriptional inducibility can also be incorporated into the expression vector. It will also be understood that the required regulatory sequences may be supplied by native proteins and / or their adjacent regions. In fact, in some embodiments, it is preferable to utilize native regulatory sequences (e.g., promoters) associated with TCR expression in organisms that have obtained a TCR.

[0105] Recombinant expression vectors may also include selection marker genes to facilitate the selection of host cells transformed or transfected with a TCR, CAR, or soluble peptide that selectively binds to Hormad1 as disclosed herein. Examples of selection marker genes include genes encoding proteins that confer resistance to certain drugs such as G418 and hygromycin, β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. The transcription of selection marker genes is monitored by changes in the concentration of selection marker proteins such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selection marker gene encodes a protein that confers antibiotic resistance, such as neomycin resistance, cells transformed with G418 (geneticin) can be selected; therefore, upon exposure to the antibiotic, cells incorporating the selection marker gene will survive while other cells will die. This makes it possible to visualize the expression of recombinant expression vectors, assay their expression, and determine the effects of mutations on expression and phenotype.

[0106] Recombinant expression vectors can be introduced into host cells to produce transformed host cells. The term “transformed host cells” is intended to include prokaryotic and eukaryotic cells transformed or transfected with the recombinant expression vectors of the present invention. The terms “transformed with,” “transfected with,” “transformation,” and “transfection” encompass the introduction of nucleic acids (e.g., vectors) into cells by one of the many possible techniques known in the art. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of this disclosure may be expressed in bacterial cells such as Escherichia coli (E. coli), insect cells (using baculoviruses), yeast cells, or mammalian cells.

[0107] The nucleic acid molecules of this disclosure can also be chemically synthesized using standard techniques. Various methods for the chemical synthesis of polydeoxynucleotides are known, including automated solid-phase synthesis using commercially available DNA synthesizers, as well as peptide synthesis (see, for example, U.S. Patents 4,598,049; 4,458,066; 4,401,796; and 4,373,071).

[0108] III. Peptide Vaccines In several aspects, methods are provided for the treatment of cancer (e.g., breast cancer, lung cancer, etc.), including a step of immunizing a target with purified tumor antigen or an immunodominant tumor antigen-specific peptide such as Hormad1 peptide (SEQ ID NO: 5). Hormad1 peptide can be administered to mammalian subjects, such as human patients, via various routes (e.g., intramuscular, intravenous, subcutaneous, etc.). In some embodiments, the peptide can be injected in a solution (e.g., saline solution) as a vaccine or to induce an immune response to the peptide. Adjuvants can be included in the formulation or solution to enhance the solubility of the peptide and / or to increase the immune response in the subject (e.g., as described in Massarelli et al., 2019). Peptide-pulsed mature dendritic cells can be administered to the subject in some embodiments. Approaches that can be used to induce an immune response or anti-cancer response to the peptide in the subject include, for example, those described in Wen et al. (2019) and Massarelli et al. (2019). In some embodiments, the Hormad1 peptide (SEQ ID NO:5) is conjugated to or presented by autologous dendritic cells that can be reinjected into a subject or human patient.

[0109] IV. Anti-cancer therapy Aspects of this disclosure relate to the administration of further anticancer therapies. In some aspects, further anticancer therapies are described herein. Examples of further anticancer therapies are provided below.

[0110] A. Immunostimulants In some embodiments, the method further comprises the administration of a further agent. In some embodiments, the further agent is an immunostimulant. As used herein, the term “immunostimulant” means a compound that can stimulate an immune response in a subject, and may include adjuvants. In some embodiments, the immunostimulant is an agent that does not constitute a specific antigen but can enhance the intensity and longevity of the immune response to an antigen. Such immunostimulants include pattern recognition receptor activators such as Toll-like receptors, RIG-1, and NOD-like receptors (NLRs); inorganic salts such as alum, alum combined with monophosphoryl lipid (MPL) A from intestinal bacteria such as Escherichia coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri, or especially alum combined with MPL® (ASO4), and alum combined separately with MPL A from the above bacteria; saponins such as QS-21, Quil-A, ISCOM, and ISCOMATRIX; MF59, montanide, ISA 51, and ISA 720; and AS02. This may include, but is not limited to, emulsions such as (QS21 + squalene + MPL), liposomes and liposome preparations such as AS01, synthesized or specially prepared microparticles and microcarriers such as outer membrane vesicles (OMVs) derived from bacteria such as Neisseria gonorrhoeae and Chlamydia trachomatis, or depot-forming agents such as chitosan particles and Pluronic block copolymers, specially modified or prepared peptides such as muramyl dipeptides, aminoalkylglucosaminide 4-phosphates such as RC529, or proteins such as bacterial toxoids or toxin fragments.

[0111] In some embodiments, further agents include agonists for pattern recognition receptors (PRRs), specifically including but not limited to TLRs 2, 3, 4, 5, 7, 8, 9 and / or combinations thereof. In some embodiments, further agents include agonists for Toll-like receptor 3, agonists for Toll-like receptors 7 and 8, or agonists for Toll-like receptor 9; preferably, the cited immunostimulants include imidazoquinoline; e.g., R848; adenine derivatives, e.g., disclosed in U.S. Patent No. 6,329,381, U.S. Published Patent Application No. 2010 / 0075995, or WO 2010 / 018132; immunostimulatory DNA; or immunostimulatory RNA.In some aspects, further agents may also be, but are not limited to, dsRNA, polyI:C or polyI:polyC12U (available as Ampligen®, both polyI:C and polyI:polyC12U are known as TLR3 stimulants), and / or 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" WO 2008033432 A2; 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., These may include immunostimulatory RNA molecules such as those disclosed in "Modified oligoribonucleotide analogs with enhanced immunostimulatory activity" U.S. Patent Application Publication No. US 2006241076; G. Lipford et al., "Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections" WO 2005097993 A2; and G. Lipford et al., "Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screening methods" WO 2003086280 A2.In some embodiments, further agents may be TLR-4 agonists such as bacterial lipopolysaccharide (LPS), VSV-G, and / or HMGB-1. In some embodiments, further agents may include, but are not limited to, TLR-5 agonists such as flagellin, or parts or derivatives thereof, including those disclosed in U.S. Patent Nos. 6,130,082, 6,585,980, and 7,192,725.

[0112] In some embodiments, further agents may be pro-inflammatory stimuli released from necrotic cells (e.g., urate crystals). In some embodiments, further agents may be activated components of the complement cascade (e.g., CD21, CD35, etc.). In some embodiments, further agents may be activated components of immune complexes. Further agents also include complement receptor agonists, such as molecules that bind to CD21 or CD35. In some embodiments, complement receptor agonists induce endogenous complement opsonization of synthetic nanocarriers. In some embodiments, immunostimulants are cytokines, which are small proteins or biological factors (ranging from 5 kD to 20 kD) released by cells that exert specific effects on intercellular interactions, signaling, and the behavior of other cells. In some embodiments, cytokine receptor agonists are small molecules, antibodies, fusion proteins, or aptamers.

[0113] B. Immunotherapy In some aspects, further treatments include cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated as IO) is the use of the immune system to treat cancer. Immunotherapy can be classified as active, passive, or hybrid (active and passive). These approaches take advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); these are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy instructs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Immunotherapy is well known in the art, and some are described below.

[0114] 1. Inhibition of co-stimulatory molecules In some embodiments, immunotherapy includes inhibitors of costimulatory molecules. In some embodiments, the inhibitors include inhibitors 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.

[0115] 2. Dendritic cell therapy Dendritic cell therapy induces an antitumor response by having dendritic cells present tumor antigens to lymphocytes, thereby activating the lymphocytes and stimulating them to kill other cells presenting the antigens. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, dendritic cells help target cancer antigens. One example of dendritic cell-based cell carcinoma therapy is cyplücel-T.

[0116] One method to induce dendritic cells to present tumor antigens is by vaccinating them with autologous oncolytics or short peptides (small portions of proteins corresponding to protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to enhance the immune and antitumor response. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0117] Dendritic cells can also be activated in vivo by inducing GM-CSF expression in tumor cells. This can be achieved by genetically modifying tumor cells to produce GM-CSF, or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0118] Another strategy involves removing dendritic cells from the patient's blood and activating them outside the body. The dendritic cells are activated in the presence of a tumor antigen, which can be a single tumor-specific peptide / protein or tumor cell lysate (a solution of destroyed tumor cells). These cells (with a selective adjuvant) are then injected to trigger an immune response.

[0119] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies, inducing dendritic cells to mature and providing immunity against tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8, or CD40 are used as antibody targets.

[0120] 3. CAR-T cell therapy Chimeric antigen receptors (also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors, CARs) are engineered receptors that combine immune cells with novel specificity to target cancer cells. Typically, these receptors transfer the specificity of monoclonal antibodies to T cells. The receptors are called chimeric because parts from different sources are fused together. CAR-T cell therapy refers to treatments that use such transformed cells for cancer treatment.

[0121] The fundamental principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activation functions. The general premise of CAR-T cells is to artificially create T cells that are designed to target markers found on cancer cells. Scientists can remove T cells from a person, genetically modify them, and return them to the patient to attack cancer cells. Once a T cell is engineered to become a CAR-T cell, it acts as a "living drug." CAR-T cells create a link between an extracellular ligand-recognition domain and an intracellular signaling molecule, which activates the T cell. The extracellular ligand-recognition domain is typically a single-stranded variable fragment (scFv). A critical aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells, and not normal cells, are targeted. The specificity of CAR-T cells is determined by the selection of the molecules being targeted.

[0122] Exemplary CAR-T therapies include tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta). In some embodiments, CAR-T therapies target CD19.

[0123] 4. Cytokine therapy Cytokines are proteins produced by many types of cells present within tumors. They can modulate the immune response. Tumors often utilize cytokines to promote tumor growth and reduce the immune response. These immunomodulatory effects make it possible to use them as drugs to induce an immune response. Two commonly used cytokines are interferons and interleukins.

[0124] Interferons are produced by the immune system. They are typically involved in antiviral responses, but are also used in cancer treatment. They are classified into three groups: Type I (IFNα and IFNβ), Type II (IFNγ), and Type III (IFNλ).

[0125] Interleukins possess numerous immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0126] 5. Adoptive T cell therapy Adoptive T-cell therapy is a form of passive immunity through T-cell transfusion (adoptive cell transfer). T cells are found in blood and tissues and are normally activated when they encounter foreign pathogens. Specifically, T cells are activated when their surface receptors encounter cells that present a portion of a foreign protein on a surface antigen. These can be either infected cells or antigen-presenting cells (APCs). They are found in normal tissues and tumor tissues, in which case they are known as tumor-infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. These cells can attack tumors, but the intratumor environment is highly immunosuppressive, preventing immune-mediated tumor death.

[0127] Multiple methods have been developed to produce and obtain tumor-targeted T cells. Tumor antigen-specific T cells can be removed from tumor samples (TILs) or filtered from blood. Subsequent activation and culture are performed ex vivo, and the cells are then reinjected. Activation can be achieved through gene therapy or by exposing T cells to tumor antigens.

[0128] 6. Checkpoint inhibitors and concomitant treatments In some embodiments, further treatments include immune checkpoint inhibitors. Certain embodiments are described further below.

[0129] PD-1 can act in the tumor microenvironment where T cells encounter infection or tumors. Activated T cells upregulate PD-1, and continue to express PD-1 in peripheral tissues. Cytokines such as IFN-gamma induce PDL1 expression in epithelial and tumor cells. PDL2 is expressed in macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive tissue damage during the immune response. The inhibitors of this disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0130] 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.

[0131] In some embodiments, a PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain contexts, 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 ligand-binding partner. In certain contexts, 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 ligand-binding partner. In certain contexts, 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. Patents 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art as described in U.S. Patent Publications US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0132] 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 the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin containing the extracellular portion or PD-1 binding moiety of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PDL1 inhibitor includes AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and 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 PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Further PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0133] In some embodiments, immune checkpoint inhibitors are PDL1 inhibitors such as durvalumab, also known as MEDI4736; atezolizumab, also known as MPDL3280A; avelumab, also known as MSB00010118C; MDX-1105; BMS-936559; or combinations thereof. In certain aspects, immune checkpoint inhibitors are PDL2 inhibitors such as rHIgM12B7.

[0134] In some embodiments, the inhibitor comprises the heavy and light chain CDR or VR 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, as well as the CDR1, CDR2, and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding to the same epitopes on PD-1, PDL1, or PDL2 as the aforementioned antibody, and / or binds to the same epitopes on PD-1, PDL1, or PDL2 as the aforementioned antibody. In another embodiment, the antibody has variable region amino acid sequence identity with the aforementioned antibody of at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or within a derivable range).

[0135] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to 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 inhibitory signals to T cells. CTLA4 is analogous 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 inhibitory signals to T cells, while CD28 transmits stimulating signals. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. When T cells are activated via the T cell receptor and CD28, the expression of CTLA-4, an inhibitory receptor for the B7 molecule, increases. The inhibitors of this disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0136] In some embodiments, immune checkpoint inhibitors are anti-CTLA-4 antibodies (e.g., human antibodies, humanized antibodies, or chimeric antibodies), their antigen-binding fragments, immunoadhesins, fusion proteins, or oligopeptides.

[0137] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the method of the present invention can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; formerly also known as ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998 can be used in the method disclosed herein. The disclosures of each of the aforementioned publications are incorporated herein 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 Nos. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0138] Further anti-CTLA-4 antibodies useful as checkpoint inhibitors in the methods and compositions of this disclosure are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or its antigen-binding fragments and variants (see, for example, WO01 / 14424).

[0139] In some embodiments, the inhibitor comprises the heavy and light chain CDR or VR 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 binding to the same epitopes on PD-1, B7-1, or B7-2 as the aforementioned antibody, and / or binds to the same epitopes on PD-1, B7-1, or B7-2 as the aforementioned antibody. In another embodiment, the antibody has variable region amino acid sequence identity with the aforementioned antibody of at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or within a derivable range).

[0140] C. Oncolytic viruses In some aspects, further treatments involve oncolytic viruses. Oncolytic viruses are viruses that selectively infect cancer cells and kill them. Once 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 not only cause the direct destruction of tumor cells but also stimulate the host's anti-tumor immune response for long-term immunotherapy.

[0141] D. Polysaccharides In some aspects, further therapeutic approaches involve polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can upregulate the immune system and may possess anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophages, NK cells, T cells, and immune system cytokines, and are being investigated in clinical trials as immunological adjuvants.

[0142] E. Newly generated antigens In some aspects, further treatments involve the administration of nascent antigens. Many tumors express mutations. These mutations potentially generate new, targetable antigens (nascent antigens) for use in T-cell immunotherapy. The presence of CD8+ T cells in cancerous lesions, identified using RNA sequencing data, is elevated in tumors with high mutational burdens. Levels of transcripts related to the cytolytic activity of natural killer cells and T cells are positively correlated with mutational burdens in many human tumors.

[0143] F. Chemotherapy In some embodiments, further treatments include chemotherapy. Suitable classes of chemotherapeutic agents include: (a) alkylating agents, e.g., nitrogen mustards (e.g., mechloretamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethyleneimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin), and triazines (e.g., dicarbazine); (b) antimetabolites, e.g., folate analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine), and purine analogs and related substances (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); (c) The present invention comprises natural products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-α), as well as (d) various activating agents (miscellaneous agents), such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrecocortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0144] Cisplatin has been widely used to treat cancers 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 therefore must be delivered via 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, effective doses used in clinical applications are intended, including approximately 15 mg / m2 to approximately 20 mg / m2 over 5 days every 3 weeks for a total of 3 processes. In some embodiments, the amount of cisplatin delivered to cells and / or subjects in combination with a construct containing an Egr-1 promoter functionally linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount that would be delivered if cisplatin were used alone.

[0145] Other suitable chemotherapeutic agents include antimicrotubule agents, such as paclitaxel ("Taxol") and doxorubicin hydrochloride ("Doxorubicin"). The combination of the Egr-1 promoter / TNFα construct delivered via an adenovirus vector and doxorubicin has been shown to be effective in overcoming resistance to chemotherapy and / or TNF-α, suggesting that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-α.

[0146] Doxorubicin is poorly absorbed and is preferably administered intravenously. In certain embodiments, an appropriate intravenous dose for adults includes approximately 60 mg / m2 to 75 mg / m2 at intervals of approximately 21 days, or approximately 25 mg / m2 to 30 mg / m2 on each of two or three consecutive days, repeated at intervals of approximately three to four weeks, or approximately 20 mg / m2 once a week. The lowest dose should be used in elderly patients if there is prior myelosuppression or neoplastic myeloinfiltration caused by prior chemotherapy, or if the drug is combined with other myelopoiopioid suppressants.

[0147] Nitrogen mustard is another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustard may include, but is not limited to, mechloretamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolicin), and chlorambucil. Cyclophosphamide (CYTOXAN® is available from Mead Johnson, and NEOSTAR® is available from Adria) is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous doses include, for example, initially in divided doses of about 40 mg / kg to about 50 mg / kg over about 2 to about 5 days, or about 10 mg / kg to about 15 mg / kg every 7 to about 10 days, or twice weekly, about 3 mg / kg to about 5 mg / kg, or about 1.5 mg / kg to about 3 mg / kg / day. Due to potential adverse effects on the gastrointestinal tract, intravenous administration is preferred. Drugs may also be administered intramuscularly, by infiltration, or into body cavities.

[0148] Further suitable chemotherapeutic agents include pyrimidine analogs such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU), and floxuridine (fluorodeoxyuridine; FudR). 5-FU may be administered to subjects at doses ranging from approximately 7.5 to approximately 1000 mg / m2. Furthermore, the 5-FU dosing schedule may vary in duration, for example, up to 6 weeks, or may be determined by those skilled in the art to whom this disclosure relates.

[0149] Another suitable chemotherapeutic agent, gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., "gemcitabine"), is recommended for the treatment of advanced and metastatic pancreatic cancer and therefore would also be useful in this disclosure for these cancers.

[0150] The amount of chemotherapeutic agent delivered to a patient may be variable. In one suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause cancer arrest or regression in the host when the chemotherapy is administered with a construct. In another embodiment, the chemotherapeutic agent may be administered in an amount somewhere between 2 and 10,000 times less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount about 20 times less, about 500 times less, or even about 5,000 times less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutic agents of this disclosure can be tested in vivo for the desired therapeutic activity in combination with a construct and for determining an effective dose. For example, such compounds can be tested in a suitable animal model system, including but not limited to rats, mice, chickens, cattle, monkeys, and rabbits, before testing in humans. As described in the examples, in vitro testing can also be used to determine suitable combinations and doses.

[0151] G. Radiation therapy In some embodiments, further or prior treatments include radiation, such as ionizing radiation. As used herein, “ionizing radiation” means radiation that has sufficient energy or that includes particles or photons capable of producing sufficient energy through nuclear interactions to produce ionization (gain or loss of electrons). Exemplary and preferred ionizing radiation is X-rays. Means for delivering X-rays to target tissue or cells are well known in the art.

[0152] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in a single 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 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 that can be derived within that range), at most 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 within which can be derived), 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 within which can be derived). In some embodiments, ionizing radiation is administered in doses of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times (or any range within which it can be derived), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times (or any range within which it can be derived), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times (or any range within which it can be derived). If more than one dose is administered, the doses may be spaced approximately 1, 4, 8, 12, or 24 hours apart, or 1, 2, 3, 4, 5, 6, 7, or 8 days apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any range within which it can be derived.

[0153] In some embodiments, the amount of IR may be presented as the total dose of IR, which is 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 50-90 Gy, administered in 20-60 fractional doses of 2-3 Gy each. According to some sources, the total dose of IR is at least 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 that can be derived within that range), at most 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 that can be derived within that range), or approximately 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 within which can be derived). In some embodiments, the total dose is administered in fractional doses of at least 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), at most 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), 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). According to some accounts, at least 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, 7 0, 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 times (or any range that can be derived within that), at most 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 times (or any range that can be derived within that), 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, A fractional dose of 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 within which it can be derived) is administered. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 fractional doses (or any range derivable therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 fractional doses (or any range derivable therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 fractional doses (or any range derivable therein) are administered per day. In some embodiments, at least 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 times (or any range that can be derived therein), at most 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 times (or any range that can be derived therein), 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 fractional doses (or any range within that range) are administered per week.

[0154] H. Surgery Approximately 60% of people with cancer undergo some form of surgery, including prophylactic surgery, surgery for diagnosis or staging, radical surgery, and palliative surgery. Radical surgery involves excision in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies such as the treatments of the present invention, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor excision refers to the physical removal of at least part of the tumor. In addition to tumor excision, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs procedure).

[0155] When cancer cells, tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment may be carried out by perfusing, directly injecting, or topically applying further anticancer therapy to the area. Such treatment may 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 may also be administered in varying doses.

[0156] I. Other active substances To improve the therapeutic efficacy of the treatment, other active agents may be used in combination with certain aspects of the embodiments of the present invention. These further active agents include those that affect the upregulation of cell surface receptors and gap junctions, cell division arresting and differentiation agents, cell adhesion inhibitors, agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological active agents. Increasing the number of gap junctions thereby increasing intercellular signaling enhances the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cell division arresting or differentiation agents may be used in combination with certain aspects of the embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are intended to improve the efficacy of the embodiments of the present invention. Examples of cell adhesion inhibitors are local adhesion kinase (FAK) inhibitors and lovastatin. To further improve the efficacy of the treatment, other active agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the embodiments of the present invention.

[0157] V. Protein Compositions As used herein, “protein,” “peptide,” or “polypeptide” refers to a molecule containing at least five amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that is naturally present in an organism. In some embodiments, the wild-type version of a protein or polypeptide is used; however, in many embodiments of this disclosure, a modified protein or polypeptide is used to produce an immune response. The above terms may be used interchangeably. “Modified protein,” “modified polypeptide,” or “variant” refers to a protein or polypeptide whose chemical structure, in particular its amino acid sequence, has been altered from that of a wild-type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). A modified / variant protein or polypeptide may be modified with respect to one activity or function, but is particularly intended to retain the wild-type activity or function in other respects, such as immunogenicity.

[0158] Where a protein is specifically referred to herein, it generally refers to a natural (wild-type) or recombinant (modified) protein, or optionally, a protein from which any signal sequence has been removed. Proteins may be isolated directly from organisms in which they are natural, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides (e.g., antibodies or fragments thereof). The term “recombinant” may be used in combination with the name of a polypeptide or a particular polypeptide, but it generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is a replication product of such a molecule.

[0159] In certain embodiments, the size of a peptide, protein, or polypeptide (wild-type or modified), such as the peptide or protein of this disclosure including the peptide of SEQ ID NO: 5, or the TCR configuration of SEQ ID NO: 2, 4, 6-11, 13, or 15, is at least 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, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any range deriveable therefrom, at most 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, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, and 2500 amino acid residues. or more, and any range that can be derived within that, or approximately 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 ,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,275,300,325,350,375,40 Polypeptides may include, but are not limited to, 0, 425, 450, 475, 500, 525, 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 derivable therein. Polypeptides may be mutated by truncation, becoming shorter than their corresponding wild-type form, and may be modified by fusing or conjugating heterologous protein or polypeptide sequences with specific functions (e.g., for targeting or localization, for enhancing immunogenicity, for purification purposes, etc.).

[0160] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of this disclosure are at least 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 from there) or more, at most 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 from there) (or any range within which can be derived) or more, 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 within which can be derived) or more, or about 1, May contain 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) or more variant amino acids or nucleic acid substitutions, or SEQ ID NO: 1-15, at least 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, or at most 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, 75 A sequence of 0, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 consecutive amino acids or nucleic acids may be similar, identical, or homologous to 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 that can be derived therein). In certain embodiments,Peptides or polypeptides are those that do not exist in nature, and / or are combinations of peptides or polypeptides.

[0161] In some embodiments, a protein or polypeptide or nucleic acid is a single amino acid or nucleic acid of SEQ ID NO: 1-15, 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, 7 7, 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,The peptides may include 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, or 300 (or any range within which can be derived). In some embodiments, the peptides of this disclosure are SEQ ID NO: 2, 4, 5 ~ 11, 13 or 15, one of 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,Carboxy of peptides containing or consisting of 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 consecutive amino acids At least 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 deriveable therefrom), at most 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 that can be derived from there), approximately 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, It includes 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range that can be derived from them), 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 that can be derived from them).

[0162] In some embodiments, proteins, polypeptides, or nucleic acids are SEQ ID NO: At least one of 2, 4, 5 to 11, 13, or 15: 1, 2, 3, 4, 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 (or any range that can be derived from there), at most 1, 2, 3, 44, 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, 6 4, 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, 10 4, 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, 1 98, 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, 22 9, 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 (or any range that can be derived from them), exactly 1, 2, 3, 44, 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, 1 67, 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, 19 8, 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 (or any range that can be derived from there), or approximately 1, 2, 3, 44, 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, 1 35, 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, 1 66, 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, 19 7, 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,It may contain 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, or 270 consecutive amino acids (or any range within that range that can be derived).

[0163] In some embodiments, polypeptides, proteins, or nucleic acids are one of SEQ ID NO: 1-15 and 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 that can be derived within that range), at most 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% SEQ IDs that are similar, identical, or homologous by 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 within that) NO: May contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or any range derivable from there), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or any range derivable from there), 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 derivable from there) consecutive peptide amino acids or nucleic acids.

[0164] In some situations, SEQ ID NO: Position numbers from 1 to 15: 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, 8 0, 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 ,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,14 6, 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 ,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,2 38, 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,SEQ ID starting with 269 or 270 NO: At least one of 1-15: 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, 8 0, 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, 1 46, 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 (or any range that can be derived from them), at most 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, 7 4, 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, 1 43, 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, 1 74, 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, 20 5, 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 (or any range that can be derived from them), 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 ,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,11 1, 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, 2 05, 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, 23 6, 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,There are polypeptides, nucleic acids (or nucleic acid molecules encoding such polypeptides) containing 268, 269, or 270 consecutive amino acids (or any range derivable within that range).

[0165] The compositions of this disclosure are intended to contain approximately 0.001 mg to approximately 10 mg of total polypeptides, peptides, and / or proteins per 1 ml. The protein concentrations in the compositions are approximately 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, and 10.0. mg / ml or higher (or any derivable range within that), at least approximately 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 It can be mg / ml or more (or any range within which it can be derived) 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 within which it can be derived).

[0166] The following is a consideration of altering the amino acid subunits of a protein to produce equivalent or, in some cases, improved second-generation variant polypeptides or peptides. For example, certain amino acids may be used in place of other amino acids in a protein or polypeptide sequence, with or without a significant loss of interaction and binding ability with structures such as the antigen-binding region of an antibody or a binding site on a substrate molecule. Since the interaction ability and properties of a protein determine its functional activity, specific amino acid substitutions can be made in the protein sequence and in its corresponding DNA coding sequence, while still producing a protein with similar or desirable properties. Thus, the inventors intend that various changes can be made in the DNA sequence of a protein-coding gene without significantly losing its biological utility or activity.

[0167] The term “functionally equivalent codon” is used herein to refer to codons that code for the same amino acid, such as the six different codons for arginine. “Neutral substitution” or “neutral mutation,” which refers to a change in a codon that codes for a biologically equivalent amino acid, is also considered.

[0168] The amino acid sequence variants of the Disclosure may be substitutional, insertional, or deletion variants. Mutations of the polypeptides of the Disclosure may 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 discontinuous or consecutive amino acids in the protein or polypeptide compared to the wild type (or any range within which such can be derived). A variant may include an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90% identical to any sequence provided or referred to herein, including all values ​​and ranges in between. A variant may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substituted amino acids.

[0169] It is understood that amino acid and nucleic acid sequences may each contain additional residues, such as further N-terminal or C-terminal amino acids, or 5' or 3' sequences, as long as they meet the above criteria, including the maintenance of biological protein activity in which protein expression is involved, and yet may still be essentially identical as described in one of the sequences disclosed herein. Terminal additions are particularly applicable to nucleic acid sequences and may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0170] Deletion variants typically lack one or more residues from the natural or wild-type protein. Individual residues may be deleted, or several adjacent amino acids may be deleted. A cleaved protein can be created by introducing a stop codon (by substitution or insertion) into the encoding nucleic acid sequence.

[0171] Insertion mutants typically involve the addition of amino acid residues at non-terminal points in a polypeptide. This can include the insertion of one or more amino acid residues. Terminal adducts can also be constructed, and these may include fusion proteins that are polymers or chains of one or more peptides or polypeptides described or referenced herein.

[0172] Substitutional variants typically involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide, and may be designed to modify one or more properties of the polypeptide, with or without loss of other functions or properties. Substitutions may be conservative, meaning one amino acid may be replaced with an amino acid having similar chemical properties. “Conservative amino acid substitutions” may involve the exchange of one amino acid class member with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, 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 may include amino acid residues that do not exist in nature and are usually incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptide mimetic molecules or other inverted or reversed amino acid moieties.

[0173] Alternatively, the substitution may be “non-conservative” in such a way that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting one amino acid residue with a chemically different residue, such as using a polar or charged amino acid in place of a non-polar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member from one amino acid class with a member from another class.

[0174] Those skilled in the art can determine appropriate variants of polypeptides, such as those described herein, using well-known techniques. Those skilled in the art can identify appropriate regions of a molecule that can be modified without disrupting its activity by targeting regions not considered important for activity. Those skilled in the art can also identify conserved amino acid residues and molecular regions among similar proteins or polypeptides. In a further embodiment, regions that may be important for biological activity or structure may undergo conserved amino acid substitutions without significantly altering biological activity or adversely affecting the protein or polypeptide structure.

[0175] When making such changes, the hydroxyl index of amino acids may be taken into consideration. The hydroxyl profile of a protein is calculated by assigning a numerical value ("hydroxyl index") to each amino acid and then repeatedly averaging these values ​​along the peptide chain. Each amino acid is assigned a value based on its hydrophobic and charge properties. These 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of hydroxyl amino acid indices in conferring interactive biological functions to proteins is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). The relative hydroxyl characteristics of amino acids contribute to the resulting secondary structure of proteins or polypeptides, and as a result, this is accepted to define the interaction between proteins or polypeptides and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It is also known that certain amino acids can be substituted with other amino acids having similar hydroxyl indices or scores, and still retain similar biological activity. When making changes based on hydroxyl indices, in certain embodiments, substitutions of amino acids with hydroxyl indices within ±2 are included. In some aspects of the present invention, substitutions within ±1 are included, and in other aspects of the present invention, substitutions within ±0.5 are included.

[0176] It is also understood in the art that similar amino acids can be effectively substituted based on their hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein governed by the hydrophilicity of adjacent amino acids correlates with the biological properties of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein governed by the hydrophilicity of adjacent amino acids correlates with its immunogenicity and antigen binding, i.e., with the biological properties of the protein. The following hydrophilic values ​​have been assigned to these amino acid residues: Arginine (+3.0); Lysine (+3.0); Aspartic acid (+3.0±1); Glutamic acid (+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). When modifications are made based on similar hydrophilicity values, in some embodiments, substitutions of amino acids with hydrophilicity values ​​within ±2 are included; in other embodiments, those within ±1 are included; and in yet another embodiment, those within ±0.5 are included. In some cases, epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also called “epitope core regions.” It is understood that amino acids can be substituted with other amino acids that have similar hydrophilicity values, and still result in biologically and immunologically equivalent proteins.

[0177] Furthermore, those skilled in the art can rethink structure-function studies to identify residues in similar polypeptides or proteins that are important for their activity or structure. From such comparative standpoints, the importance of amino acid residues in proteins corresponding to amino acid residues important for the activity or structure of similar proteins can be predicted. Those skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0178] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to its structure in similar proteins or polypeptides. Considering such information, those skilled in the art can predict the alignment of amino acid residues of the polypeptide with respect to its three-dimensional structure. Those skilled in the art may choose not to alter amino acid residues predicted to be on the protein surface, as such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art can create test variants containing a single amino acid substitution at the position of each desired amino acid residue. These variants can then be screened using standard assay methods for binding and / or activity, thus obtaining information gathered from such routine experiments, which allows those skilled in the art to determine amino acid positions where further substitution should be avoided, either alone or in combination with other mutations. Various tools available for determining secondary structure can be found on the World Wide Web at expasy.org / proteomics / protein_structure.

[0179] In some embodiments of the present invention, amino acid substitutions are made to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) modify binding affinity for protein complex formation, (4) modify ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, one or more amino acid substitutions (conservative amino acid substitutions in certain embodiments) may be made in naturally occurring sequences. Substitutions may be made in the portion of an antibody that is outside the domain that forms an intermolecular contact. In such embodiments, conservative amino acid substitutions that do not substantially alter the structural features of the protein or polypeptide (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes the natural antibody) may be used.

[0180] VI. Pharmaceutical preparations In selected embodiments, a Hormad1-derived peptide (e.g., SEQ ID NO: 5), cells expressing a TCR disclosed herein (e.g., any of SEQ ID NO: 1-4) (e.g., T cells), or a protein containing a variable region of a TCR disclosed herein is intended to be administered to a subject to induce a therapeutic immune response against cancer (e.g., a solid tumor expressing Hormad1). A pharmaceutical composition for use in a subject may comprise a TCR disclosed herein, such as a soluble TCR (which may optionally be attached to a contrast agent or therapeutic agent) or a bispecific TCR, and a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition may contain further immunostimulatory compounds or anticancer agents.

[0181] The terms “pharmaceutical,” “pharmaceutically acceptable,” or “pharmacologically acceptable” mean, as appropriate, molecular entities and compositions that, when administered to animals, such as humans, do not cause adverse reactions, allergic reactions, or other adverse reactions. As used herein, “pharmaceutically acceptable carrier” includes, as is well known to those skilled in the art, all kinds of solvents, dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and such similar materials and combinations thereof (e.g., Remington: The Science and Practice of Pharmacy, 22, incorporated herein by reference). nd See edition, Pharmaceutical Press, 2012. Unless any conventional carrier is incompatible with the proteins (e.g., Hormad1 peptide, soluble TCR) or cells (e.g., T cells expressing the TCR) of the present disclosure, the vaccine composition or adoptive cell transfer therapy of the present invention is intended for use.

[0182] As used herein, “therapeutic immune response” or “defensive immune response” refers to the response of the mammalian host’s immune system to cancer. A defensive immune response may provide therapeutic effects for the treatment of cancer, such as a reduction in tumor size or improved survival.

[0183] Those with ordinary skills in the medical technology field will understand that the actual dosage of a therapeutic composition administered to an animal or human patient may be determined by physical and physiological factors such as body weight, severity of condition, type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and route of administration. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.

[0184] The therapeutic compositions disclosed herein may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intralesionally, intracerebrally, intraarticularly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosa, intrapericardially, intraocularly, orally, topically, locally, or by injection, infusion, continuous infusion, lavage, and local perfusion. The therapeutic compositions may also be administered to a target via a catheter, in a lipid composition, or by other means or any combination thereof, as is known to those skilled in the art (e.g., Remington: The Science and Practice of Pharmacy, 22, incorporated herein by reference). nd See Ed., Pharmaceutical Press, 2012.

[0185] Any suitable carrier known to those skilled in the art may be used in the pharmaceutical compositions of the present invention, although the type of carrier will vary depending on the mode of administration. For parenteral administration, such as intravenous, intratumoral, or subcutaneous injection, the carrier may include water, saline, alcohol, fat, wax, or buffer. Biodegradable microspheres (e.g., polylactic acid galactide) may also be used as carriers in some embodiments. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.

[0186] In some embodiments, vaccine compositions may be administered by microstructured transdermal or ballistic particle delivery. Microstructures as carriers for vaccine formulations are desirable configurations for vaccine applications and are widely known in the art (e.g., U.S. Patents No. 5,797,898, 5,770,219 and 5,783,208, and U.S. Patent Application No. 2005 / 0065463). Microstructures or ballistic particles that function as support substrates for TCRs, such as soluble TCRs, disclosed herein may consist of biodegradable and non-biodegradable materials, and such support substrates may consist of synthetic polymers, silica, lipids, carbohydrates, proteins, lectins, ionic agents, crosslinking agents, and other microstructure components available in the art. Protocols and reagents for immobilizing the peptides of the present invention onto support substrates composed of such materials are widely available commercially.

[0187] In other embodiments, the vaccine composition comprises an immobilized or encapsulated TCR or soluble TCR and a support substrate as disclosed herein. The support substrate may include, but is 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 nanocarriers and microcarrier formulations are generally known to those skilled in the art, and the use of liposomes, microparticles, nanocapsules, etc., is widespread in the delivery of therapeutic substances (e.g., U.S. Patent No. 5,741,516, which is incorporated specifically in whole by reference). Numerous methods of liposomes and liposome-like preparations as potential drug carriers, including peptide encapsulation, are known and can be used in various embodiments (e.g., U.S. Patents No. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0188] In any case, the composition may contain various antioxidants to slow the oxidation of one or more components. Furthermore, prevention of microbial action can be achieved by preservatives such as various antimicrobial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0189] The composition must be stable under manufacturing and storage conditions and must be protected from contamination by microorganisms such as bacteria and fungi. It will be understood that endotoxin contamination should be kept to a minimum at a safe level, for example, less than 0.5 ng / mg of protein.

[0190] A. Combination therapy In certain embodiments, compositions and methods of this embodiment, 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, α-β T cells, or γ-δ T cells), NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), or induced pluripotent stem (iPS) cells), may be administered to a mammalian subject (e.g., human) in combination with at least one further therapy. The further therapy may be radiotherapy, surgery (e.g., primary surgery, tumor removal, mammary gland tumor removal, or mastectomy), chemotherapy, conditioning chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. The further therapy may take the form of adjuvant therapy or neoadjuvant therapy.

[0191] In some embodiments, further therapy is the administration of small molecule enzyme inhibitors or anti-metastatic agents. In some embodiments, further therapy is the administration of one or more side effect limiting agents (e.g., antiemetics, or other agents that can mitigate the occurrence and / or severity of side effects of the procedure). In some embodiments, further therapy is radiotherapy. In some embodiments, further therapy is surgery. In some embodiments, further therapy is a combination of radiotherapy and surgery. In some embodiments, further therapy is gamma irradiation. In some embodiments, further therapy is chemotherapy, such as dacarbazine or temozolomide. Further therapy may be one or more chemotherapeutic agents known in the art.

[0192] T-cell therapy or adoptive cell transfer therapy can be administered before, during, or after further cancer therapies, such as immune checkpoint therapy or conditioning chemotherapy, or in various combinations. Such administrations can occur at intervals ranging from simultaneous to minutes, days, or weeks. In embodiments where T-cell therapy is delivered to the patient separately from further therapeutic agents, it will generally be necessary to ensure that the two compounds do not expire between each delivery time so that they can still exert a beneficial combined effect for the patient. In such cases, antibody therapy and anti-cancer therapy may be delivered to the patient within approximately 12–24 or 72 hours of each other, and especially within approximately 6–12 hours of each other. In some situations, it may be desirable to significantly extend the treatment period if several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) pass between each administration.

[0193] Various combinations can be used. In the following example, antigen-specific T-cell therapy, peptides, or TCRs are "A", and anticancer therapy is "B". TIFF2026053605000010.tif18128

[0194] The administration of any compound or therapy in this embodiment to a patient will follow general protocols for administering such compounds, taking into account the toxicity of the drug, if any. Therefore, in some embodiments, there will be a stage for monitoring toxicity resulting from the combination therapy. [Examples]

[0195] VII. Examples The following embodiments are included to demonstrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments demonstrate that the techniques discovered by the inventors are fully functional in the practice of the present invention and therefore constitute a preferred mode for that practice. However, in consideration of this disclosure, those skilled in the art will understand that many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, and similar or equivalent results can still be obtained.

[0196] Example 1 Hormad1-specific T cell receptors redirect T cells towards tumor cells. To further explore the potential of the Hormad1 T cell epitope as a therapeutic target for clinical immunotherapy, full-length Hormad1-56 TCR α and β chains were inserted into the retroviral vector pMSGV3, and then PBMCs were infected using this recombinant retroviral vector (Figure 3). An empty retroviral vector was used as a control. After infection, CD8+ / tetramer+ populations were observed by FCM detection. After tetramer induction, sorting, and expansion, high-purity TCR-T cells were generated. Hormad1 overexpression was observed in the tumor tissue of approximately 50% of non-small cell lung cancer (NSCLC) patients tested, and elevated Hormad1 expression was not observed in healthy tissue (Hormad1 is not expressed in healthy tissue other than the testes). High Hormad1 expression correlated with increased mutational burden in the lung adenocarcinoma patient population (Nichols et al., 2018), but it remained unclear whether this protein could be used as an immunotherapy target.

[0197] TCR-transduced T cells specifically recognized T2 cells pulsed with Hormad1 peptide with high binding affinity and lysed HLA-A2+ Hormad1-expressing tumor cell lines, but did not lyse Hormad1-, HLA-A2-, or normal cells (Figure 4). Hormad1-specific TCR-transduced T cells could recognize these solid tumor cells but could not recognize control tumor cells (Figure 4). These results suggest that Hormad1-derived peptides are expressed in association with HLA-A2 molecules on tumor cells, and that Hormad1-TCR-transduced T cells can be used for cancer immunotherapy.

[0198] Hormad1-56 TCR-T Functional Assay To further explore the function of Hormad1-56 TCR-T, 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 was co-cultured with several tumor cell lines. When co-cultured with HLA-A2+ Hormad1-expressing tumor cell lines, the levels of CD137, CD69, TNF-α, and IFN-γ expressed by TCR-T were significantly enhanced, but this was not observed in antigen-negative cells, similar to control tumor cell lines (Figure 5).

[0199] The TCR sequence was derived from the parent Hormad1-56 CTL cell line A12. The Hormad1-56 TCR (hereinafter referred to as Hormad1-TCR) is TRAV4 * 01 F, TRBV13 * 01 F and TRAV4 * 01 F, TRBV13 * It was revealed that the sequence is composed of the 01 F 2 subfamily (Figure 6).

[0200] Example 2 material and method Healthy donor PBMC sample This study was approved by the institutional review board at the University of Texas MD Anderson Cancer Center. Informed consent was obtained in accordance with the Declaration of Helsinki before collecting PBMC samples from healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by leukocyte separation.

[0201] cell line T2 hybridoma cells, lung cancer cell lines H1395, H522, H1299, H1299-A2, H1355, H1755, DFC1032, K562-A2, K562-A2-eGFP, K562-A2-Hormad1, H522-eGFP, and H522-Hormad1 were cultured at 37°C and in 5% CO2 air in RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 1×Glutamax, 50 μM β-mercaptoethanol, 1 mM sodium pyruvate, 100 U / mL penicillin + 100 μg / mL streptomycin, and 10 μg / mL gentamicin (all from Invitrogen, Carlsbad, CA). Normal lung cell line HSAEC2-KT was cultured in serum-free Small Airway Epithelial Cell Growth Medium (PromoCell, Heidelberg, Germany).

[0202] Isolation of tumor and immune cell subsets CD25- T cells were isolated by magnetic cell separation (MACS, Miltenyi Biotec, Auburn, CA), and their purity was confirmed by flow analysis. This procedure yielded CD25- T cells with a purity of over 90%.

[0203] reagent Mouse anti-human antibodies against CD3, CD4, CD8, CD69, CD137, IFN-γ, and TFN-α were obtained from Biolegend, San Diego, CA. All peptides were synthesized to over 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.

[0204] PCR Total RNA was extracted from T cells using the RNeasy Kit (Qiagen). Approximately 3 μg of total RNA was reverse transcribed into cDNA using the SMARTer® RACE 5' / 3' Kit (ClonTech). TCR fragments were cloned by PCR using the Q5® High-Fidelity 2X Master Mix Kit (NEB) under the following conditions: 40 cycles of 2 minutes at 98°C, followed by 15 seconds at 98°C, 30 seconds at 63°C, and 45 seconds at 72°C using the Bio-Rad PCR System. The PCR products were cloned into pRACE vectors using the In Fusion Cloning Kit (ClonTech), and then DNA sequencing was performed using BigDye® Direct Cycle Sequencing Kits (Thermo).

[0205] Flow cytometry For intracellular staining, cells were fixed and permeabilized using the Fixation / Permeabilization Kit (eBioscience) according to the manufacturer's instructions. The cells were then stained with mouse anti-human flow antibody (as described above; 00114) at 4°C for 30 minutes. After two washes, the samples were acquired using FACS Calibur (BD Biosciences) and analyzed using Cell Quest 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 Hormad1 tetramer and APC-Cy7-conjugated mouse anti-human CD8 antibody were mixed with cells in 50 μl volume at room temperature for 30 minutes, washed twice, and analyzed by flow cytometry.

[0206] Creation of Hormad1-56 peptide-specific CTL strains Mature DCs derived from HLA-A0201+ healthy donors were pulsed with Hormad1-56 peptide (YLDDLCVKI; SEQ ID NO: 5) and stimulated with autologous CD25- T cells. After two rounds of stimulation, Hormad1-56 specific T cell lines were detected and sorted using the corresponding Hormad1-56 tetramer and anti-CD8 antibody. CD8+ / tetramer+ T cells were expanded using a rapid expansion protocol (REP), and the purity of Hormad1-56 specific T cells was determined by anti-CD8 antibody and tetramer staining.

[0207] Generation of Hormad1-specific TCR-T cells using retroviruses The complete TCRαβ sequence of the Hormad1 T cell line was obtained by 5-RACE RT-PCR and codon-optimized. The constant regions of the α and β chains were cysteine-mutated; the TCRαβ chains were ligated with furin and P2A and cloned into retroviral vectors. Retroviruses containing the TCR were produced in 293 T cells, filtered, concentrated, and stored at -80°C. HLA-A2+ healthy donor T cells were activated with OKT3 antibody and IL-2 for 72 hours, transduction with retrovirus was performed in a 2000 g centrifuge at 32°C for 2 hours, followed by overnight incubation. After 48 hours, antigen-specific TCR expression was analyzed by tetramer staining. Tetramer-positive T cells were selected by flow and further expanded by REP for further functional assays as described previously (Pollack et al., 2014).

[0208] Cell damage assay T2 cells were pulsed with gradually decreasing peptide concentrations (10 μg / ml to 10 pg / ml) and used as targets in a standard 4-hour Cr51-releasing cytotoxicity assay. Tumor cell lines (2 × 10) 3 Cells (100 cells / well) were incubated with effector T cells in a 96-well round-bottom plate at 37°C for 4 hours at the indicated ratio (E:T = 40:1 to 1.25:1), and target cell lysis was determined by a Cr51 release assay. All assays were performed in triple wells and repeated at least twice.

[0209] statistical analysis Student's t-test was used to compare various experimental groups. A p-value < 0.05 was considered statistically significant. Unless otherwise specified, the mean and standard deviation are shown.

[0210] All methods disclosed and asserted herein can be prepared and carried out without excessive experimentation, given the present invention. While the compositions and methods of the present invention have been described in preferred embodiments, it will be apparent to those skilled in the art that the methods and the steps or order of steps described herein can be modified without departing from the concept, spirit, and scope of the present invention. More specifically, certain chemically and physiologically related agents can be used instead of the agents described herein, and it will be apparent that the same or similar results can be obtained at the same time. All such similar substitutions and modifications, which will be apparent to those skilled in the art, are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0211] References The following references are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement what is described herein. TIFF2026053605000011.tif201153TIFF2026053605000012.tif238159TIFF2026053605000013.tif209158

Claims

[Claim 1] The invention described in the specification of this application.

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