MANA Body and Method of Use

Abstract

To provide methods and materials for assessing a mammal having or suspected of having cancer and / or for treating a mammal having cancer.SOLUTION: The invention provides molecules comprising an antigen-binding domain that can bind to a peptide-HLA-beta-2 microglobulin complex, where the peptide comprises a modified peptide, where the HLA is a class I HLA, and where the antigen-binding domain does not bind to a complex that includes a wild-type version of the modified peptide. Also provided are methods of using such molecules.SELECTED DRAWING: Figure 1

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of U.S. Patent Application No. 62 / 506,674, filed May 16, 2017 The disclosure of the prior application is considered to be part of the disclosure of this application (and is incorporated by reference into the disclosure of this application).

[0002] (Statement Regarding Federal Government Funds) This invention was made with government support under grant number CA62924 from the National Institutes of Health The United States government has certain rights in this invention.

Technical Field

[0003] (1. Technical Field) This document relates to methods and materials for evaluating a mammal having or suspected of having cancer and / or for treating a mammal having cancer. For example this document provides methods and materials for treating a mammal having cancer using a molecule comprising one or more antigen - binding domains (e.g., single - chain variable fragments (scFv)) that can bind to a modified peptide (e.g., a tumor antigen).

Background Art

[0004] (2. Background Information) Somatic mutations in cancer are ideal targets for cancer therapy because they are specifically expressed only in tumor cells and not in normal cells. In particular there are advantages to targeting driver gene proteins (broadly subdivided into oncogene proteins and tumor suppressor proteins). First resistance is less likely to occur due to dependence on the tumorigenic ability of the tumor. Second these mutations usually occur early in tumor growth, so essentially all daughter cancer cells contain the mutation ​​ will occur. Finally, driver gene proteins tend to have hotspot mutations shared among many patients, so treatment methods targeting a single mutation can be applied to a wide range of patient populations. Most mutant proteins, including most mutant driver gene proteins, are intracellular. Small molecules can target intracellular proteins, but the development of small molecules that can specifically inhibit the activity of mutant driver genes rather than their wild-type (wt) counterparts has remained out of reach for most such driver gene proteins. Antibodies that can distinguish single amino acid mutations can typically only target extracellular epitopes.

[0005] SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] The immune system samples the intracellular contents of cells through antigen processing and presentation. Following proteolysis, some of the resulting peptides are loaded onto human leukocyte antigen (HLA) and sent to the cell surface, where they function for T cells to distinguish self-peptides from non-self peptides via the T cell receptor (TCR). For example, virus-infected cells present viral peptides on their HLA and cause T cells to kill the cells. Similarly, in cancer, mutant peptides are presented on HLA on the surface of cancer cells, which is abbreviated as Mutation-Associated Neo-Antigen or MANA. In some cases, to varying degrees, patients have these mutant peptide-HLA may initiate an anti-cancer T cell response against neoantigens, and checkpoint blockade antibodies further enhance this response. However, many patients, especially those with low mutational burdens, are unable to initiate a sufficient anti-cancer T cell response. Therefore, a therapy or diagnostic method that specifically targets MANA can provide a truly tumor-specific approach for diagnosing or treating cancer.

[0007] HLA class I proteins are present on all nucleated cells. There are three classical HLA class I genes, A, B, and C, each of which is highly polymorphic. Each HLA allele has a specific peptide-binding motif, such that only specific peptides will bind to a particular HLA allele.

[0008] In the art, there continues to be a need to develop new methods for diagnosing, monitoring, and effectively treating cancer.

[0009] This document provides methods and materials for treating a mammal having cancer. For example, this document provides methods and materials using one or more molecules comprising one or more antigen-binding domains (e.g., scFv ) that can bind to a modified peptide (e.g., a modified peptide present in a peptide-HLA-b2M complex) to treat a mammal having cancer (e.g., a cancer that expresses the modified peptide). Optionally, one or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to a modified peptide (e.g., a modified peptide present in a peptide-HLA-b2M complex) are administered to a mammal having cancer (e.g., a cancer that expresses the modified peptide), and thereby treating the mammal. ​​​​Mammals can be treated.

Means for Solving the Problems

[0010] As demonstrated herein, single-chain variable fragments (scFvs) that target multiple mutation-associated neoantigens (MANAs) present in the peptide-HLA-b2M complex in many acute myeloid leukemia (AML) cases (e.g., bind to them) have been identified. Also, as demonstrated herein, using the scFvs, both chimeric antigen receptor (CAR) T cells (CART; also abbreviated as CAR T or CAR-T) and bispecific antibodies that can recognize and kill cells expressing MANA have been designed. The ability to specifically target MANA provides tumor-specific methods for diagnosing and / or treating cancer. For example, an scFv that specifically targets MANA can be used in a full-length antibody or a fragment thereof, an antibody-drug conjugate (ADC), an antibody-radionuclide conjugate, CART, or a bispecific antibody to diagnose and / or treat mammals having cancer. targeting multiple mutation-associated neoantigens (MANAs) present in the peptide-HLA-b2M complex (e.g., bind to) have been identified. Also, as demonstrated herein, using the scFvs, chimeric antigen receptor (CAR) T cells (CART; CAR T or also abbreviated as CAR-T) and bispecific antibodies that can recognize and kill cells expressing MANA have been designed. The ability to specifically target MANA provides tumor-specific methods for diagnosing and / or treating cancer. For example, an scFv that specifically targets MANA can be used in a full-length antibody or a fragment thereof, an antibody-drug conjugate (ADC), an antibody-radionuclide conjugate, CART, or a bispecific antibody to diagnose and / or treat mammals having cancer. and / or treating cancer. For example, an scFv that specifically targets MANA is used in a full-length antibody or a fragment thereof, an antibody-drug conjugate (ADC), an antibody-radionuclide conjugate, CART, or a bispecific antibody to diagnose and / or treat mammals having cancer. mammals having cancer can be diagnosed and / or treated.

[0011] Generally, one aspect of the present document is a molecule comprising an antigen-binding domain capable of binding to a peptide-HLA-beta-2 microglobulin complex, wherein the peptide comprises a modified peptide, the HLA is class I HLA, and the antigen-binding domain does not bind to a complex comprising the wild-type version of the modified peptide. The modified peptide may comprise 7 to 15 amino acids (e.g., 10 amino acids). The modified peptide is a modified IDH2 polypeptide, a modified EGFR polypeptide, a modified p53 polypeptide, a modified KRAS polypeptide, a modified HRAS polypeptide, a modified NRAS polypeptide, or a modified CTNNB polypeptide. capable of binding to a peptide-HLA-beta-2 microglobulin complex, wherein the peptide comprises a modified peptide, the HLA is class I HLA, and the antigen-binding domain does not bind to a complex comprising the wild-type version of the modified peptide. The modified peptide may comprise 7 to 15 amino acids (e.g., 10 amino acids). The modified peptide is a modified IDH2 polypeptide, a modified EGFR polypeptide, a modified p53 polypeptide, a modified KRAS polypeptide, a modified HRAS polypeptide, a modified NRAS polypeptide, or a modified CTNNB polypeptide. The modified peptide may comprise 7 to 15 amino acids (e.g., 10 amino acids). The modified peptide may be a modified IDH2 polypeptide, a modified EGFR polypeptide, a modified p53 polypeptide, a modified KRAS polypeptide, a modified HRAS polypeptide, a modified NRAS polypeptide, or a modified CTNNB polypeptide. a modified IDH2 polypeptide, a modified EGFR polypeptide, a modified p53 polypeptide, a modified KRAS polypeptide, a modified HRAS polypeptide, a modified NRAS polypeptide, or a modified CTNNB polypeptide HRAS polypeptide, a modified NRAS polypeptide, or a modified CTNNB polypeptide may be derived from. The modified peptide may contain the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 1 5, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID se quence NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 3 2. When the modified peptide contains SEQ ID NO: 1, class I HLA may be HLA-B7, and the antigen-binding fragment may contain the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ quence NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. When the modified peptide contains SEQ ID NO: 11, class I HLA may be HLA-B7, and the antigen-binding fragment may contain the amino acid sequence shown in SEQ ID NO: 380, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, or SEQ ID NO: 393. When the modified peptide contains SEQ ID NO: 13, class I HLA may be HLA-A2, and the antigen-binding fragment may contain the amino acid sequence shown in SEQ ID NO: 3 24, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 3 29, or SEQ ID NO: 330. When the modified peptide contains SEQ ID NO: 15, class I HLA may be HLA-A2, and the antigen-binding fragment may be the amino acid sequence shown in SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 336, or SEQ ID NO: 337. When the modified peptide contains SEQ ID NO: 16, class I HLA may be H LA-A2, and the antigen-binding fragment may contain the amino acid sequence shown in SEQ ID NO: 332, SEQ ID NO: 334, SEQ ID se quence NO: 335, SEQ ID NO: 336, or SEQ ID NO: 337. When the modified peptide contains SEQ ID NO: 18, class I HLA may be HLA-A2, and the anti gen-binding fragment may contain the amino acid sequence shown in SEQ ID NO: 338, SEQ ID NO: 339, or SEQ ID NO: 340 When the modified peptide contains SEQ ID NO: 18, class I HLA may be HLA-A2, and the antigen-binding fragment may contain the amino acid sequence shown in SEQ ID NO: 338, SEQ ID NO: 339, or SEQ ID NO: 340 LA-A2, and the antigen-binding fragment may contain the amino acid sequence shown in SEQ ID NO: 332, SEQ ID NO: 334, SEQ ID se quence NO: 335, SEQ ID NO: 336, or SEQ ID NO: 337. When the modified peptide contains SEQ ID NO: 18, class I HLA may be HLA-A2, and the anti gen-binding fragment may contain the amino acid sequence shown in SEQ ID NO: 338, SEQ ID NO: 339, or SEQ ID NO: 340 LA-A2, and the antigen-binding fragment may contain the amino acid sequence shown in SEQ ID NO: 338, SEQ ID NO: 339, or SEQ ID NO: 340 may include the amino acid sequence. When the modified peptide includes SEQ ID NO: 20, class I HLA may be HLA-A3, and the antigen-binding fragment may include the amino acid sequence represented by SEQ ID NO: 341, SEQ ID NO: 342, or SEQ ID NO: 343. When the modified peptide includes SEQ ID NO: 21, class I HLA may be HLA-A3, and the antigen-binding fragment may include SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, or the amino acid sequence represented by SEQ ID NO: 357. When the modified peptide includes SEQ ID NO: 22, class I HLA may be HLA-A3, and the antigen-binding fragment may include SEQ ID NO: 338 , SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343 , SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348 , SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373 , or the amino acid sequence represented by SEQ ID NO: 374. When the modified peptide includes SEQ ID NO: 2 4, class I HLA may be HLA-A11, and the antigen-binding fragment may include SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, or the amino acid sequence represented by SEQ ID NO: 368. When the modified peptide includes SEQ ID NO: 26 , class I HLA may be HLA-A3, and the antigen-binding fragment may include SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, or the amino acid sequence represented by SEQ ID NO: 379 It may contain the amino acid sequence to be obtained. When the modified peptide contains SEQ ID NO: 28, class I H LA can be HLA-A1, and the antigen-binding fragment may contain the amino acid sequence represented by SEQ ID NO: 394 acid sequence. When the modified peptide contains SEQ ID NO: 30, class I HLA is HLA -A1, and the antigen-binding fragment may contain the amino acid sequence represented by SEQ ID NO: 395 When the modified peptide contains SEQ ID NO: 31, class I HLA can be HLA-A1 and the antigen-binding fragment may contain the amino acid sequence represented by SEQ ID NO: 396. When the modified pe ptide contains SEQ ID NO: 32, class I HLA can be HLA-A1, and the antigen-binding fragment may contain the amino acid sequence represented by SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, or the amino acid sequence represented by SEQ ID NO: 401. The molecule can be an antibody, antibody fragment tor, scFv, CAR, TCR, TCR mimic, tandem scFv, bispecific T cell e ngager, diabody, single-chain diabody, scFv-Fc, bispecific antibody, or bispecific affinity retargeting antibody (DART). For example, the molecule can be a single-chain diab ody. The molecule may also contain an antigen-binding domain that can bind to an effector cell receptor (e.g., CD3, CD28, CD4 , CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, or CD27). The antigen-binding domain that can bind to an effector cell may also be able to bind to CD3 In this case, the antigen-binding domain is SEQ ID NO: 404, SEQ ID NO: 405, SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 411, SEQ ID NO: 412, SEQ ID NO: 413, SEQ ID NO: 414, SEQ ID NO: 415, SEQ ID NO: 416, or SEQ When binding to 3, the antigen-binding domain is SEQ ID NO: 404, SEQ ID NO: 405, SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 411, SEQ ID NO: 412, SEQ ID NO: 413, SEQ ID NO: 414, SEQ ID NO: 415, SEQ ID NO: 416, or SEQ It may contain the amino acid sequence shown by column number 417.

[0012] In another aspect, this document features a CAR. The CAR binds to any antigen described herein A binding domain (e.g., an antigen-binding domain capable of binding to a peptide-HLA-beta-2 microglobulin complex, where the peptide includes a modified peptide, HLA is class I HLA, and the antigen-binding domain does not bind to a complex containing the wild-type version of the modified peptide) and may include an extracellular domain, a transmembrane domain, and an intracellular domain. The transmembrane domain may include the transmembrane domain of CD4, CD8, or CD28. The intracellular domain may include one or more co-stimulatory domains from CD28, DAP10, ICOS, OX 40, and / or 4-1BB. The intracellular domain may include a signaling domain from CD3-zeta.

[0013] In another aspect, this document features T cells that express any CAR described herein (e.g., an extracellular domain, a transmembrane domain, and an intracellular domain including any antigen-binding domain described herein). The T cells have an extracellular domain including an antigen-binding domain capable of binding to a peptide-HLA-beta- 2 microglobulin complex, where the peptide includes a modified peptide, HLA is class I HLA, and the antigen-binding domain does not bind to a complex containing the wild-type version of the modified peptide), a transmembrane domain, and an intracellular domain.

[0014] In another aspect, this document features a method for treating a mammal having cancer. This method​​​​​ comprises administering to a mammal one or more molecules comprising any antigen-binding domain described herein (e.g., an antigen-binding domain capable of binding to a peptide-HLA-beta-2 microglobulin complex, wherein the peptide comprises a modified peptide, the HLA is class I HLA, and the antigen-binding domain does not bind to a complex comprising the wild-type version of the modified peptide), where the cancer comprises cancer cells expressing the modified peptide. The mammal can be human. The cancer can be Hodgkin's lymphoma, non-Hodgkin's lymphoma, AML, lung cancer, pancreatic cancer , gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, breast cancer, prostate cancer, esophageal cancer, gastric cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, or astrocytoma. In another aspect, the present document features a method of treating a mammal having cancer. The method comprises administering to a mammal one or more T cells expressing any one of the CARs described herein (e.g., a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising any antigen-binding domain described herein), where the cancer comprises cancer cells expressing the modified peptide. The mammal can be human. The cancer can be Hodgkin's lymphoma, non-Hodgkin's lymphoma, AML, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, breast cancer, prostate cancer, esophageal cancer, gastric cancer, kidney cancer, bone cancer, soft tissue cancer, head

[0015] and neck cancer, glioblastoma multiforme, or astrocytoma. The method can comprise, or consist essentially of, administering to a mammal one or more T cells expressing any one of the CARs described herein (e.g., a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising any antigen-binding domain described herein), where the cancer comprises cancer cells expressing the modified peptide. The mammal can be human. The cancer can be Hodgkin's lymphoma, non-Hodgkin's lymphoma, AML, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, breast cancer, prostate cancer, esophageal cancer, gastric cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, or astrocytoma. lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, breast cancer, prostate cancer, esophageal cancer, gastric cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, or astrocytoma.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0017] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

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DETAILED DESCRIPTION OF THE INVENTION

[0019] This document provides methods and materials for evaluating mammals having or suspected of having cancer, and / or for treating mammals having cancer. For example, one or more modified peptides (e.g., peptides present in peptide-HLA-b2M complexes such as peptide-HLA complexes) can be targeted (e.g., bound) by one or more antigen-binding domains (e.g., scFv) contained in one or more molecules to evaluate mammals having or suspected of having cancer, and / or to treat mammals having cancer (e.g., cancer expressing one or more modified peptides). Optionally, one or more molecules contain one or more antigen-binding domains that can bind to the modified peptide, and these can be used to detect the presence or absence of one or more modified peptides in a sample obtained from a mammal suffering from or suspected of having cancer. Optionally, one or more antigen-binding domains that can bind to the modified peptide are contained in one or more molecules, and these can be used to detect the presence or absence of one or more modified peptides in a sample obtained from a mammal suffering from or suspected of having cancer. Optionally, one or more antigen-binding domains that can bind to the modified peptide are contained in one or more molecules, and these can be used to detect the presence or absence of one or more modified peptides in a sample obtained from a mammal suffering from or suspected of having cancer. Optionally, one or more antigen-binding domains that can bind to the modified peptide are contained in one or more Administer a plurality of molecules to a mammal having cancer (e.g., cancer expressing a modified peptide) and the mammal can be treated.

[0020] As used herein, a modified peptide is a peptide derived from a modified polypeptide. The modified polypeptide can be any suitable modified polypeptide (e.g., a polypeptide having a mutation that causes a disease such as an oncogenic mutation). The modified peptide can have one or more amino acid modifications (e.g., substitutions) relative to the wild-type (wt) peptide (e.g., the peptide derived from the wt polypeptide from which the modified polypeptide is derived). The modified peptide can also be referred to as a mutant peptide. In some cases, the modified peptide can be a tumor antigen. Examples of tumor antigens include, without limitation, mutation-associated neoantigens (MANA), tumor-associated antigens, and tumor-specific antigens. The modified peptide can be of any suitable length. In some cases, the modified peptide can be from about 7 amino acids to about 15 amino acids (e.g., from about 8 amino acids to about 15 amino acids, from about 9 amino acids to about 15 amino acids, from about 10 amino acids to about 15 amino acids , from about 11 amino acids to about 15 amino acids, from about 12 amino acids to about 15 amino acids, from about 13 amino acids to about 15 amino acids, from about 7 amino acids to about 14 amino acids, from about 7 amino acids to about 13 amino acids, from about 7 amino acids to about 12 amino acids, from about 7 amino acids to about 11 amino acids, from about 7 amino acids to about 10 amino acids , from about 7 amino acids to about 9 amino acids, or from about 9 amino acids to about 10 amino acids) in length. For example, the modified peptide can be about 9 amino acids in length. For example, the modified peptide can be about 10 amino acids in length. The modified peptide can be derived from any modified (e.g., oncogenic) polypeptide. The modified polypeptide from which the modified peptides described herein can be derived can be any suitable modified polypeptide (e.g., a polypeptide having a mutation that causes a disease such as an oncogenic mutation). The modified peptide can have one or more amino acid modifications (e.g., substitutions) relative to the wild-type (wt) peptide (e.g., the peptide derived from the wt polypeptide from which the modified polypeptide is derived). The modified peptide can also be referred to as a mutant peptide. In some cases, the modified peptide can be a tumor antigen. Examples of tumor antigens include, without limitation, mutation-associated neoantigens (MANA), tumor-associated antigens, and tumor-specific antigens. The modified peptide can be of any suitable length. can be any modified (e.g., oncogenic) polypeptide. Examples of peptides include epidermal growth factor receptor (EGFR), isocitrate dehydrogenase 2 (IDH2), p53, RAS (e.g., KRAS, HRAS, and NRAS), and CTNNB, but are not limited thereto. Modified peptides can include any suitable modification. In some cases, the modified peptides described herein can include one or more modifications (e.g., mutations) shown in Table 1. [Table 1]

[0021] The modified peptides described herein (e.g., modified peptides comprising the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 2 2, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) can be in a complex with any suitable HLA. The HLA can be any suitable HLA allele and can be. In some cases, the HLA can be a class I HLA (e.g., HLA-A, HLA-B, H LA-C) allele. Examples of HLA alleles with which the modified peptides described herein can complex include HLA-A1, HLA-A2, HLA-A3, HLA-11, and HLA-B7, but are not limited thereto. Exemplary HLA alleles for specific modified peptides are shown in Table 1. For example, a modified peptide (e.g., IMQLMPFGC (SEQ ID NO: 13)) derived from a modified EGFR polypeptide can be in a complex with HLA-A2 and b2M. For example, a modified peptide (e.g., from a modified IDH2 polypeptide can be in a complex with HLA-A2 and b2M. For example, a modified peptide (e.g., IMQLMPFGC (SEQ ID NO: 13)) derived from a modified EGFR polypeptide can be in a complex with HLA-A2 and b2M. For example, a modified peptide derived from a modified IDH2 polypeptide (e.g., then SPNGTIQNIL (SEQ ID NO: 1) can be in a complex with HLA-B7 and b2M For example, a modified peptide derived from a modified p53 polypeptide (e.g., GMN QRPILTI (SEQ ID NO: 15) or GMNWRPILTI 1 (SEQ ID NO: 16)) can be in a complex with HLA-A2 and b2M. For example, a modified peptide derived from a modified KRAS polypeptide (e.g., LVVVGAVGV (SEQ ID NO: 18), VVVGA CGVGK (SEQ ID NO: 20), VVVVGADGVGK (SEQ ID NO: 21), VVVVGAVG VGK (SEQ ID NO: 22), and VVGADGVGK (SEQ ID NO: 24)) can be in a complex with HLA-A 2, HLA-A3, and / or HLA-A11, and b2M. For example, a modified peptide derived from a modified CTNNB polypeptide (e.g., TTAPF LSGK (SEQ ID NO: 26)) can be in a complex with HLA-A3 and b2M. For example a modified peptide derived from a modified KRAS polypeptide (e.g., AVGVGKSAL (SEQ ID NO: 11)) can be in a complex with HLA-B7 and b2M. For example, a modified peptide derived from a modified H / K / N RAS polypeptide (e.g., ILDTAGHE EY (SEQ ID NO: 28), ILDTAGKEEY (SEQ ID NO: 30), ILDTAGLEEY (SEQ ID NO: 31), ILDTAGREEY (SEQ ID NO: 32)) can be in a complex with HLA-A1 and b 2M. (SEQ ID NO: 31), ILDTAGREEY (SEQ ID NO: 32)) can be in a complex with HLA-A1 and b 2M.

[0022] This document relates to the modified peptides described herein (e.g., SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 3 1. and a modified peptide comprising an amino acid sequence represented by any one of SEQ ID NO: 32) Provided are molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to In some cases, a molecule comprising one or more antigen-binding domains that can bind to the modified peptides described herein does not target (e.g., does not bind to) the modified peptides described herein that are not present in a complex (e.g., a peptide-HLA- b2M complex). In some cases, a molecule comprising one or more antigen-binding domains that can bind to the modified peptides described herein does not target (e.g., does not bind to) a wt peptide (e.g., a peptide derived from the wt polypeptide from which the modified polypeptide is derived).

[0023] A molecule comprising one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein can be of any suitable type. In some cases, the molecule can be a monovalent molecule (e.g., comprising a single antigen-binding domain). In some cases, the molecule can be a multivalent molecule (e.g., comprising two or more antigen-binding domains and targeting two or more antigens simultaneously). For example, a bispecific molecule can comprise two antigen-binding domains, a trispecific molecule can comprise three antigen-binding domains, a tetravalent molecule can comprise four antigen-binding domains, and so on. Examples of molecules comprising antigen-binding domains include antibodies, antibody fragments, scFv, CAR, T cell receptors (T CR), TCR mimics, tandem scFv, bispecific T cell engagers, diabodies, scDb, scFv-Fc, bispecific antibodies, dual-affinity retargeting antibodies (DART ​​​​​ ) and any other molecule comprising at least one variable heavy chain (VH) and at least one variable light chain (VL), but not limited thereto. Any of these molecules can be used according to the materials and methods described herein. In some cases, the antigen-binding domain can be a scFv. For example, a molecule comprising one or more antigen-binding domains (e.g., one or more scFvs) that can bind to the modified peptides described herein can be a CAR. For example, a molecule comprising two scFvs that can bind to the modified peptides described herein can be a single-chain diabody (scDb). In some cases, when a molecule comprising one or more antigen-binding domains (e.g., one or more scFvs) that can bind to the modified peptides described herein (e.g., modified peptides comprising the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) is a CAR, the CAR can be any suitable CAR. The CARs provided herein can comprise an extracellular domain having at least one antigen-binding domain that can bind to the modified peptides, transmembrane domains, and intracellular domains (e.g., intracellular signaling domains such as co-stimulatory domains) described herein. The CAR can comprise any suitable extracellular domain. For example, the CAR can be SEQ ID NO: 1,

[0024] SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. ​​​​​​​​20, an amino acid sequence represented by any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32 A molecule having an antigen-binding domain capable of binding to a modified peptide containing the sequence (e.g., sc Fv) may be included. The CAR may include any suitable transmembrane domain. The transmembrane domain may be derived from any suitable polypeptide. Examples of transmembrane domains used in the CARs described herein include, but are not limited to, those of CD4, CD8 (e.g., CD8-alpha and CD8 -beta), CD28, CD3 epsilon, CD5, CD6, CD9, CD16, CD2 2, CD33, CD37, CD45, CD64, CD80, CD86, CD134, 4- 1BB, and CD154. In some cases the CARs described herein may include the CD28 transmembrane domain. The CAR may include a suitable intracellular domain. The intracellular domain may be derived from any suitable polypeptide and may include a co-stimulatory domain (e.g., a single co-stimulatory domain or multiple co-stimulatory domains). When the CAR includes multiple co-stimulatory domains, the CAR may include multiple co-stimulatory domains of the same type or multiple co-stimulatory domains of different types . The intracellular domain may include a signaling domain. Examples of intracellular domains that may be used in the CARs described herein include, but are not limited to, those of CD3 (e.g., CD3-zeta), CD28, DAP10, inducible T cell co-stimulator (ICOS), OX40, 4-1BB, CD2, C D4, CD8, CD5, CD22, DAP-12, CD22, and CD79 intracellular dom ains. The CAR may be made using any suitable method ​ It can be produced. In some cases, the CAR may also include a hinge sequence (e.g., located between the extracellular domain and the transmembrane domain). In some cases, the CAR can be made as described elsewhere (Curran et al., 2012 J. Gene Med 1 4:405-415; Kershaw et al., 2005 Nature Rev iews Immunol. 5(12):928-940; Eshhar et al ., 1993 Proc. Natl. Acad. Sci. U.S.A. 90( 2):720-724; Sadelain et al., 2009 Curr. Op in. Immunol. 21(2):215-223; WO 2015 / 14267 5; WO 2015 / 150526; and WO 2014 / 134165). Also, CARTs expressing one or more CARs are provided herein, and the CARs can target (e.g., bind to) one or more of the modified peptides described herein (e.g., CARs having two or more antigen-binding domains). Also, CARTs expressing one or more CARs are provided herein, and the CARTs can target (e.g., bind to) one or more of the modified peptides described herein. In some cases, the modified peptides described herein (e.g., amino acid sequences represented by any one of SEQ ID NO:1, SEQ ID NO:11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32) are included in the modified peptides.

[0025] ​​​​One or more antigen-binding domains (e.g., scFv) that can bind to When the molecule containing the is a multivalent molecule (e.g., a bispecific molecule), the first antigen-binding domain can bind to the modified peptide described in this specification, and the second antigen-binding domain can bind to an effector cell (e.g., an antigen present on an effector cell). Examples of effector cells include, but are not limited to, T cells, natural killer (NK) cells, natural killer T ( NKT) cells, B cells, plasma cells, macrophages, monocytes, microglia, dendritic cells, eosinophils, fibroblasts, and mast cells. Examples of antigens present on effector cells include, but are not limited to, CD3, CD4, CD8, CD28, CD16a, NK G2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, CD27, and any other effector cell surface receptor. Optionally, the molecule described in this specification can have a first antigen-binding domain that can bind to the modified peptide described in this specification, and a second antigen-binding domain that can bind to an antigen present on a T cell (e.g., CD3). Optionally, the sequence (e.g., scFv sequence) that can bind to CD3 can be as shown in Table 4. Optionally, the sequence (e.g., scFv sequence) that can bind to CD3 can be as otherwise described (e.g., Rodrigues et al., 1992 Int J Can cer Suppl. 7:45-50; Shalaby et al., 1992 J Exp Med. 175:217-25; Brischwein et al., 2 006 Mol Immunol. 43:1129-43; Li et al., 20 as described elsewhere (e.g., Rodrigues et al., 1992 Int J Can cer Suppl. 7:45-50; Shalaby et al., 1992 J Exp Med. 175:217-25; Brischwein et al., 2 006 Mol Immunol. 43:1129-43; Li et al., 20 006 Blood 108:1766-73; incorporated herein by reference in its entirety). 05 Immunology. 116:487-98;WO2012162067;U S20070065437;US20070065437;US20070065437 ;US20070065437;US20070065437;and US200700 65437 (see). In some cases, the molecules described herein are the modifications described herein A first antigen-binding domain capable of binding to a peptide, and an antigen present on NK cells ( For example, CD16a or NKG2D) and a second antigen-binding domain capable of binding to May include. By binding to both the modified peptide and the effector cell, the multivalent molecule Brings cells expressing the modified peptide (e.g., as part of the HLA complex) closer to the effector cells Allows effector cells to act on cells expressing the modified peptide This makes it possible.

[0026] In some cases, one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptides described herein (e.g., the modified peptide comprising the amino acid sequence shown by any one of SEQ ID NO: 1, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID Number 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ Number 31, and SEQ ID NO: 32) are multivalent molecules (e.g., bispecific molecules), the molecule may be of any suitable type comprising at least one VH and at least one VL. For example, VH and V L can be in any suitable orientation. In some cases, VH can be the N-terminus of VL . In some cases, VH can be the C-terminus of VL. In some cases, the amino acids of the linker Can be. In some cases, VH can be the C-terminus of VL. In some cases, the amino acids of the linker . In some cases, the amino acids of the linker The amino acid sequence can be arranged between VH and VL.

[0027] In some cases, when the bispecific molecule is a tandem scFv, the tandem scFv can have any suitable orientation. Examples of the orientation of a tandem scFv containing scFv-A and scFv-B include VLA-LL-VHA-SL-VLB-LL-VHB, VLA-LL -VHA-SL-VHB-LL-VLB, VHA-LL-VLA-SL-VLB-LL- VHB, VHA-LL-VLA-SL-VHB-LL-VLB, VLB-LL-VHB- SL-VLA-LL-VHA, VLB-LL-VHB-SL-VHA-LL-VLA, V HB-LL-VLB-SL-VLA-LL-VHA, and VHB-LL-VLB-SL -VHA-LL-VLA, but are not limited thereto, where SL is a short linker and LL is a long linker. The short linker can be about 3 amino acids to about 10 amino acids in length. The short linker can contain any suitable amino acids (e.g., glycine and serine) in any suitable combination. The long linker can be about 10 amino acids to about 25 amino acids in length. The long linker can contain any suitable amino acids (e.g., glycine and serine) in any suitable combination.

[0028] In some cases, when the bispecific molecule is a diabody, the diabody can have any suitable orientation. Examples of the orientation of a diabody containing scFv-A and scFv-B include VLA-SL-VHB and VLB-SL-VHA, VLA-SL-VLB and VHB-SL-VHA, VHA-SL-VLB and VHB-SL-VLA, VLB- SL-VHA and VLA-SL-VHB, VLB-SL-VLA and VHA-SL- VHB, and VHB-SL-VLA and VHA-SL-VLB are included, but this is not limited thereto, where SL is a short linker. The short linker can be about 3 amino acids to about 10 amino acids in length. The short linker can contain any suitable amino acids (e.g., glycine and serine) in any suitable combination.

[0029] In some cases, when the bispecific molecule is a scDb, the scDb can be in any suitable orientation ality. Examples of the orientation of scDb containing scFv-A and scFv-B include VL A-SL-VHB-LL-VLB-SL-VHA, VHA-SL-VLB-LL-VHB -SL-VLA, VLA-SL-VLB-LL-VHB-SL-VHA, VHA-SL- VHB-LL-VLB-SL-VLA, VLB-SL-VHA-LL-VLA-SL-V HB, VHB-SL-VLA-LL-VHA-SL-VLB, VLB-SL-VLA-L L-VHA-SL-VHB, and VHB-SL-VHA-LL-VLA-SL-VLB are included, but not limited thereto, where SL is a short linker and LL is a long linker er. The short linker can be about 3 amino acids to about 10 amino acids in length. The short li nker can contain any suitable amino acids (e.g., glycine and serine) in any suitable combination. The long linker can be about 10 amino acids to about 25 amino acids in length . The long linker can contain any suitable amino acids (e.g., glycine and serine) in any suitable combination.

[0030] ​​In some cases, when the bispecific molecule is scFv-Fc, the scFv-Fc can have any appropriate orientation. Examples of the orientation of scFv-Fc-A, scFv-Fc-B, and scFv-Fc containing the Fc domain include VLA-LL-VHA-hinge-Fc and VLB-LL-VHB-hinge-Fc, VHA-LL-VLA-hinge-Fc and V HB-LL-VLB-hinge-Fc, VLA-LL-VHA-hinge-Fc and VHB -LL-VLB-hinge-Fc, VHA-LL-VLA-hinge-Fc and VLB-L L-VHB-hinge-Fc, but are not limited thereto, where LL is a long linker The long linker can be about 10 amino acids to about 25 amino acids in length. The long linker can contain any appropriate amino acids (e.g., glycine and serine) in any appropriate combination. In some cases, the Fc domain of scFv-Fc contains one or more modifications that can increase heterodimerization of scFv-Fc and / or decrease homodimerization of scFv-Fc. In some cases, the Fc domain of scFv-Fc can exclude the hinge domain In some cases, the Fc domain of scFv-Fc can be at the N-terminus of the scFv and can be.

[0031] Molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein (e.g., modified peptides containing the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 2 2, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) can be any ​​may include appropriate complementarity determining regions (CDRs). For example, a molecule comprising one or more antigen-binding domains capable of binding to a modified peptide described herein may include a variable heavy chain (VH) having three VH complementarity determining regions (CDR-VH) and a variable light chain (VL) having three VL CDRs (CDR- VL). For example, a molecule capable of binding to a modified peptide derived from a modified EGFR polypeptide (e.g., IMQLMPFGC (SEQ ID NO: 13)) may include one of each of the CDRs shown below: derived from a modified EGFR polypeptide (e.g., IMQLMPFGC (SEQ ID NO: 13)) may include one of each of the CDRs shown below: derived from a modified EGFR polypeptide (e.g., IMQLMPFGC (SEQ ID NO: 13)) may include one of each of the CDRs shown below: derived from a modified EGFR polypeptide (e.g., IMQLMPFGC (SEQ ID NO: 13)) may include one of each of the CDRs shown below: CDR-VL1: QDVNTA (SEQ ID NO: 33); CDR-VL2: SAS; CDR-VL3: QQYDYAPIT (SEQ ID NO: 34), QQSPYYYLPIT (SEQ ID NO: 35), QQYYYSPVT (SEQ ID NO: 36), QQHYGNPFT (SEQ ID NO: 37), QQSYYSPPT (SEQ ID NO: 38), QQYYSYPPT (SEQ ID NO: 39) , QQYYYYPPT (SEQ ID NO: 40); CDR-VH1: GFNISWYQ (SEQ ID NO: 41), GFNVSWSY (SEQ ID NO: 42), GFNISWNQ (SEQ ID NO: 43), GFNVGYYG (SEQ ID NO: 44), G FNITSSY (SEQ ID NO: 45), GFNINSSY (SEQ ID NO: 46), GFNISTS Y (SEQ ID NO: 47); CDR-VH2: VTPYSGYT (SEQ ID NO: 48), IYGDSGYT (SEQ ID NO: 49), VSPYSGYT (SEQ ID NO: 50), VSGMEGYT (SEQ ID NO: 51), I SPADGYN (SEQ ID NO: 52), ISPTDGYY (SEQ ID NO: 53), IDPNDGY S (SEQ ID NO: 54); and CDR-VH3: SRSYTDGFDY (SEQ ID NO: 55), SRGQWEASYY AMDY (SEQ ID NO: 56), SRSDYYAMDY (SEQ ID NO: 57), SRDIYGYA MDV (SEQ ID NO: 58), SRTDSTAYTAMDV (SEQ ID NO: 59), SRTSDT SYAAMDV (SEQ ID NO: 60), SRTNNTAADAMDV (SEQ ID NO: 61).

[0032] For example, a modified peptide derived from a modified IDH2 polypeptide (e.g., SPNGTIQ A molecule that can bind to NIL (SEQ ID NO: 1) may include each of the following CDRs: One: CDR-VL1: QDVNTA (SEQ ID NO: 33); CDR-VL2: SAS; CDR-VL3: QQYSYSPPT (SEQ ID NO: 62), QQGKAYWPAT( SEQ ID NO: 63), QQVYSSPFT (SEQ ID NO: 64), QQYSLYSPMT (SEQ ID NO SEQ ID NO: 65), QQSYYMPFT (SEQ ID NO: 66); CDR-VH1: GFNISDTY (SEQ ID NO: 67), GFNVGHYR (SEQ ID NO SEQ ID NO: 68), GFNVKYYM (SEQ ID NO: 69), GFNSFLS (SEQ ID NO: 70), GF NIFRGY (SEQ ID NO: 71); CDR-VH2: ISPRTGYN (SEQ ID NO: 72), VSPNGYYT (SEQ ID NO SEQ ID NO: 73), ISPGYDYT (SEQ ID NO: 74), IFPSSDYT (SEQ ID NO: 75), I SPHSDYT (SEQ ID NO: 76); and CDR-VH3: SRAYYSYAYAMDV (SEQ ID NO: 77), SRGYSSY AFDY (SEQ ID NO: 78), SRSYWRYSVDV (SEQ ID NO: 79), SRGKHSS DSNYYMDY (SEQ ID NO: 80), SRSYGWAAFDY (SEQ ID NO: 81)

[0033] For example, a modified peptide derived from a modified p53 polypeptide (e.g., GMNQRPIL TI (SEQ ID NO: 15) and GMNWRPILTI (SEQ ID NO: 16) The resulting molecule may contain one of each of the CDRs set forth below: CDR-VL1: QDVNTA (SEQ ID NO: 33); CDR-VL2:SAS; CDR-VL3: QQSGYAPIT (SEQ ID NO: 82), QQYSYAPIT (SEQ ID NO: Column number 83), QQSLYGPFT (sequence number 84), QQYSYSPIT (sequence number 8 5), QQSGYQPDT (SEQ ID NO: 86), QQYLYQPWT (SEQ ID NO: 87); CDR-VH1: GFNISYYS (SEQ ID NO: 89), GFNIGYYT (SEQ ID NO: No. 90), GFNIAYEY (SEQ ID NO: 91), GFNLFGYG (SEQ ID NO: 92), G FNISWYA (SEQ ID NO:93), GFNIDYYG (SEQ ID NO:94); CDR-VH2: VDPDSDYT (SEQ ID NO: 96), VSPWSYST (SEQ ID NO: No. 97), IGPDSGYT (SEQ ID NO: 98), IGPYYYYT (SEQ ID NO: 99), I WPDSDWT (SEQ ID NO: 100), LYGGSDST (SEQ ID NO: 101); and CDR-VH3: SRSWIHMFSMDY (SEQ ID NO: 103), SRDHWDE AFDV (SEQ ID NO: 104), SRVWYYSTYGMDY (SEQ ID NO: 105), SRE NYDMAMDY (SEQ ID NO: 106), SRYYYSSAFDV (SEQ ID NO: 107), S RQYSAYFDY (sequence number 108).

[0034] For example, modified peptides derived from modified KRAS polypeptides (e.g., LVVVGAV GV (SEQ ID NO: 18), VVVGACGVGK (SEQ ID NO: 20), VVVGADGVGK (SEQ ID NO:21), VVVGAVGVGK (SEQ ID NO:22), and VVGADGVGK Molecules that can bind to (Accession No. 24) can each contain one of the following CDRs shown below : CDR-VL1: QDVNTA (Accession No. 33); CDR-VL2: SAS and SAY; CDR-VL3: QQWYSSPVT (Accession No. 110), QQYYSRPVT( Accession No. 111), QQSYSGSGSPWT (Accession No. 121), QQTYYSPWT( Accession No. 122), QQYYYPPIT (Accession No. 123), QQSYYYFRPIT( Accession No. 132), QQASYYYPLT (Accession No. 133), QQKSEYSPWT( Accession No. 134), QQSGYIPFT (Accession No. 135), QQGAYYRPFT( Accession No. 136), QQYMYSPVT (Accession No. 152), QQSSSSPIT(Accession No. 153), QQSSASPLT (Accession No. 154), QQYAYSPLT (Accession No. 1 55), QQYSYYPIT (Accession No. 168), QQYSYTPVT (Accession No. 169 ), QQYSYEPVT (Accession No. 170), QQYAYYSPVT (Accession No. 171) , QQYEYYPMT (Accession No. 172), QQYSFYPFT (Accession No. 188), Q QYSYSPIT (Accession No. 85), QQYSAYYQPIT (Accession No. 189), QQ YSYYPIT (Accession No. 168), QQYEYVPHT (Accession No. 190), QQYS YMPIT (Accession No. 191), QQYAYYPVT (Accession No. 192), QQYSYM PIT (Accession No. 191), QQYDYRPVT (Accession No. 193), QQYDFTPM T (Accession No. 194), QQYSSSSPVT (Accession No. 195), QQSSYTPIT (Accession No. 229), QQYAYYPIT (Accession No. 230), QQYEYYPIT( Column number 231), QQYTYYPIT (Sequence number 232), QQYSYYPIT (Sequence number 168), QQSSVEPWT (Sequence number 233); CDR-VH1: GFNINWAN (Sequence number 112), GFNIYLHD (Sequence number 113), GFNIYWSH (Sequence number 114), GFNIVGGG (Sequence number 12 4), GFNIRSYA (Sequence number 125), GFNVSHTG (Sequence number 126), G FNLSYSD (Sequence number 137), GFNISASG (Sequence number 138), GFNIY RYG (Sequence number 139), GFNIYGTM (Sequence number 140), GFNISYSY( Sequence number 141), GFNVSAYW (Sequence number 156), GFNISGYG (Sequence number 157), GFNVSSVG (Sequence number 158), GFNVSSYG (Sequence number 159) 、GFNFSYGY (Sequence number 173), GFNVMWGPG (Sequence number 174), GFN VSGSQ (Sequence number 175), GFNIYGQM (Sequence number 176), GFNVMYS T (Sequence number 177), GFNFGSY (Sequence number 196), GFNISDSY (Sequence number 197), GFNIFSDQ (Sequence number 198), GFNLSYSY (Sequence number 199 ), GFNISYGY (Sequence number 200), GFNISYQH (Sequence number 201), GF NLSGYY (Sequence number 202), GFNVSGQY (Sequence number 203), GFNVST SG (Sequence number 204), GFNISYAK (Sequence number 205), GFNFSSYV (Sequence number 206), GFNISQGG (Sequence number 234), GFNISSTG (Sequence number 2 35), GFNFFSTV (Sequence number 236), GFNLHGYL (Sequence number 237), GFNLSTHV (Sequence number 238), GFNVSYYS (Sequence number 239); CDR-VH2: ISPPYDYT (SEQ ID NO: 115), IIPAYDYT (SEQ ID NO: 116), ISSFEGYT (SEQ ID NO: 117), IYPQGDYT (SEQ ID NO: 12 7), VGPGKGYT (SEQ ID NO: 128), VGPGKGYT (SEQ ID NO: 128), V MPDSGHT (SEQ ID NO: 142), IHPLKPYT (SEQ ID NO: 143), LYPYG YST (SEQ ID NO: 144), FKPDSYNT (SEQ ID NO: 145), LLPYDGNT( SEQ ID NO: 146), IYGGSGYT (SEQ ID NO: 160), LYGGSDYT (SEQ ID NO: 161), IYGTSDYT (SEQ ID NO: 162), IAPRRDYT (SEQ ID NO: 163) 、ISGYTGNT (SEQ ID NO: 178), IHPFSGNT (SEQ ID NO: 179), IPG WSGYT (SEQ ID NO: 180), LSPFSGNT (SEQ ID NO: 181), IYSWSDY T (SEQ ID NO: 182), ISGYSGNT (SEQ ID NO: 207), FSPYSSNT (SEQ ID NO: 208), FMPYDSYYT (SEQ ID NO: 209), ISGFSGNT (SEQ ID NO: 2 10), FHYGSGNT (SEQ ID NO: 211), FMPYQGST (SEQ ID NO: 212), FSPYSGYT (SEQ ID NO: 213), ISPVSGNT (SEQ ID NO: 214), IYGA YSGT (SEQ ID NO: 215), LTYWGGYT (SEQ ID NO: 216), VYPDSGGT (SEQ ID NO: 217), VYPGGGQT (SEQ ID NO: 240), LLGGSGNT (SEQ ID NO : 241), IYPWSGST (SEQ ID NO: 242), IYPPNGYT (SEQ ID NO: 243 ), FYPYVGYT (SEQ ID NO: 244), IYPWNDYT (SEQ ID NO: 245); And CDR-VH3: SRSYSYYFDY (SEQ ID NO: 118), SRRDGYYFD Y (SEQ ID NO: 119), SRSYSYYMDY (SEQ ID NO: 120), SRDSSYLAF DY (Accession No. 129), SRNFQSTSHAFDY (Accession No. 130), SRKTY YAFDY (Accession No. 131), SRATNIPVYAFDY (Accession No. 147), SR YSSMYYYYFDY (Accession No. 148), SRSYAYGYFAY (Accession No. 149 ), SRGEVYHYYAFDY (Accession No. 150), SRAAYSSMDV (Accession No. 151), SRTHSYWSAFDY (Accession No. 164), SRTVRYAFDY (Accession No. 165), SRSSRYSMDY (Accession No. 166), SRKSSYYFDY (Accession No. 167), SRAASLSSSYYSAFDV (Accession No. 183), SRGYSYS AMDY (Accession No. 184), SRGYSYFAMDY (Accession No. 185), SRNIS YEQSSAFDY (Accession No. 186), SRGYAHNSFDY (Accession No. 187), SRSNQSAYSYMDY (Accession No. 218), SRSQFTFYQYFDY (Accession No. 219), SRMSVRNAFDY (Accession No. 220), SRSDSYYTAMDY ( Accession No. 221), SRSNYYYLDY (Accession No. 222), SRANIYSSHSF FDY (Accession No. 223), SRTHSSIYHSFDY (Accession No. 224), SRPM KTSYYGAFDY (Accession No. 225), SRSQSYTYWSAMDY (Accession No. 2 26), SRGEYGTYMDY (Accession No. 227), SRTSSYYAFDY (Accession No. 228), SRGYDYSAFDY (Accession No. 246), SRGLQYSAMDY (Accession No. 247), SRSRSSNYYFDV (Accession No. 248), SRGVDYAYLD Y (Accession No. 249), SRGYRYQYMDV (Accession No. 250), SRGSYYSF DY (Accession No. 251).

[0035] For example, a molecule that can bind to a modified peptide derived from a modified CTNNB polypeptide (e.g., TTAPFL SGK (SEQ ID NO: 26)) may contain any one of the following CDRs: : CDR-VL1: QDVNTA (SEQ ID NO: 33); CDR-VL2: SAS and SAY; CDR-VL3: QQSYYSPPT (SEQ ID NO: 38), QQIYTSPIT ( SEQ ID NO: 252), QQRAYFPIT (SEQ ID NO: 253), QQQYAYTPIT (SEQ ID NO: 254), QQIHYKPLT (SEQ ID NO: 255); CDR-VH1: GFNINNTY (SEQ ID NO: 256), GFNFITTG (SEQ ID NO: 257), GFNFSDYG (SEQ ID NO: 258), GFNVWSYG (SEQ ID NO: 25 9), GFNVAWYS (SEQ ID NO: 260); CDR-VH2: IYPTDGYT (SEQ ID NO: 260), IGPGSDYT (SEQ ID NO: 261), LIPASGYT (SEQ ID NO: 262), VTPDGSYT (SEQ ID NO: 26 3), VYGGSSYT (SEQ ID NO: 264); and CDR-VH3: SRTYYSYYSAMDV (SEQ ID NO: 265), SRYYYA SALDY (SEQ ID NO: 266), SRGWSYYMDY (SEQ ID NO: 267), SRSYG WAMDY (SEQ ID NO: 268), SRDFYSSGMDY (SEQ ID NO: 269).

[0036] For example, a molecule that can bind to a modified peptide derived from a modified KRAS polypeptide (e.g., AVGVGKS AL (SEQ ID NO: 11)) may contain any one of the following CDRs: : CDR-VL1: QDVNTA (SEQ ID NO: 33); CDR-VL2: SAS; CDR-VL3: QQEWRLPIT (SEQ ID NO: 270), QQGTSTPFT( (SEQ ID NO: 271), QQSWRYPMT (SEQ ID NO: 272), QQSYSYPVT (SEQ ID NO: 273), QQGWLYSPFT (SEQ ID NO: 274); CDR-VH1: GFNVYGNQ, (SEQ ID NO: 275), GFNLSYYG (SEQ ID NO: 402), GFNISRYG (SEQ ID NO: 276), GFNIYSSW (SEQ ID NO 2 77), GFNISGYG (SEQ ID NO: 157); CDR-VH2: IYPYSGST (SEQ ID NO: 278), IYPDSGYT (SEQ ID NO: 279), FYPSSSYT (SEQ ID NO: 280), FQPYSGYT (SEQ ID NO: 28 1), VYGGSGYT (SEQ ID NO: 282); and CDR-VH3: SRSAYVAYSYFDY (SEQ ID NO: 283), SRAYLY YYLAY (SEQ ID NO: 284), SRKYYEAMDY (SEQ ID NO: 285), SREYT YYFDY (SEQ ID NO: 286), SRAHSSYYVDY (SEQ ID NO: 287).

[0037] For example, a molecule that can bind to a modified peptide (e.g., IL DTAGHEEY (SEQ ID NO: 28), ILDTAGKEEY (SEQ ID NO: 30), ILDT AGLEEY (SEQ ID NO: 31), ILDTAGREEY (SEQ ID NO: 32)) derived from a modified H / K / N RAS polypeptide may contain one of each of the following CDRs: CDR-VL1: QDVNTA (SEQ ID NO: 33); CDR-VL2: SAS; CDR-VL3: QQHYYSPVT (SEQ ID NO: 292), QQYAYAPFT( (SEQ ID NO: 296), QQAHMIPIT (SEQ ID NO: 300), QQSVYDPIT (SEQ ID NO: 301), QQAYYSPIT (SEQ ID NO: 302); No. 301), QQSYTSPLT (SEQ ID NO: 302), QQGQYSPFT (SEQ ID NO: 303), QQYWYLPTT (SEQ ID NO: 320); CDR-VH1: GFNIGYYG (SEQ ID NO: 289), GFNIFYQD (SEQ ID NO: 293), GFNVSYSM (SEQ ID NO: 297), GFNFSFPG (SEQ ID NO: 30 5), GFNISGSW (SEQ ID NO: 306), GFNIYYGV, (SEQ ID NO: 307), GFNVSYEY (SEQ ID NO: 308), GFNISWYD (SEQ ID NO: 321); CDR-VH2: VYPGGGYT (SEQ ID NO: 290), IYPDYDYT (SEQ ID NO: 294), VWGDGGVT (SEQ ID NO: 298), FVGYDGYT (SEQ ID NO: 31 0), LYPDSDYT (SEQ ID NO: 311), IYPDSSWT (SEQ ID NO: 312), I YGGSDNT (SEQ ID NO: 313), IEPSVGYT (SEQ ID NO: 322); and CDR-VH3: SRYYYYGFDY (SEQ ID NO: 291), SRTYSVYMD Y (SEQ ID NO: 295), SRGSYYAFDY (SEQ ID NO: 299), SRDYYSFSM DY (SEQ ID NO: 316), SRAHTYAFDY (SEQ ID NO: 317), SRDQDFHY MNYYLSYALDY (SEQ ID NO: 318), SRPLGSYFDY (SEQ ID NO: 319) ), SRSYPYYYFDY (SEQ ID NO: 323).

[0038] Examples of CDRs that can bind to specific modified peptides (e.g., CDR-VL1, C DR-VL2, CDR-VL3, CDR-VH1, CDR-VH2, and CDR-VH 3 in certain combinations) are shown in Table 2. In some cases, the modified peptides described herein (e.g., SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, Any one of SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:2 6, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32 One or more antibodies that can bind to a modified peptide) comprising the amino acid sequence shown A molecule comprising an antigen-binding domain (e.g., scFv) may comprise any suitable set of CDR sequences ( e.g., any of the sets of CDR sequences described herein).

[0039] A molecule comprising one or more antigen-binding domains (e.g., scFv) that can bind to a modified peptide (e.g., a modified peptide comprising the amino acid sequence shown by any one of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:2 2, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32) may comprise any suitable sequence. For example, a molecule that can bind to a modified peptide derived from a modified EGFR polypeptide ( e.g., IMQLMPFGC (SEQ ID NO:13)) may comprise an scFv sequence shown by any one of SEQ ID NO:324, SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327, SEQ ID NO:328, SEQ ID NO: 329, and SEQ ID NO:330, but is not limited thereto. For example, a molecule that can bind to a modified peptide derived from a modified IDH2 polypeptide (e.g., SPNGTIQNIL (SEQ ID NO:1)) may comprise an scFv sequence shown by any one of SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8, but is not limited thereto. For example, a molecule that can bind to a modified p53 polypeptide may comprise an scFv sequence shown by any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8, but is not limited thereto. For example, a molecule that can bind to a modified p53 polypeptide may comprise an scFv sequence shown by any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8, but is not limited thereto. For example, a molecule that can bind to a modified p53 polypeptide e.g., SPNGTIQNIL (SEQ ID NO:1)) may comprise an scFv sequence shown by any one of SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8, but is not limited thereto. For example, a molecule that can bind to a modified p53 polypeptide may comprise an scFv sequence shown by any one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8, but is not limited thereto. For example, a molecule that can bind to a modified p53 polypeptide Molecules that can bind to the derived modified peptides (e.g., GMNQRPILTI (SEQ ID NO: 15) and GMNW RPILTI 1 (SEQ ID NO: 16)) can include, but are not limited to, scFv sequences represented by any one of SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, and may include, but are not limited to, scFv sequences represented by any one of SEQ ID NO: 337. For example, molecules that can bind to modified peptides derived from modified KRAS polypeptides (e.g., LVV VGAVGV (SEQ ID NO: 18), VVVGACGVGK (SEQ ID NO: 20), VVVGAD GVGK (SEQ ID NO: 21), VVVGAVGVGK (SEQ ID NO: 22), and VVGAD GVGK (SEQ ID NO: 24)) can include, but are not limited to, scFv sequences represented by any one of SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373, and SEQ ID NO: 374, and may include, but are not limited to, scFv sequences represented by any one of them. For example, molecules that can bind to modified peptides derived from modified CTNNB polypeptides (e.g., TTAPF LSGK (SEQ ID NO: 26)) can include, but are not limited to, s represented by any one of SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379 LSGK (SEQ ID NO: 26)) can include, but are not limited to, s represented by any one of SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379 may include, but are not limited to, cFv sequences. For example, to bind to a modified peptide (e.g., AVGVGKSAL (SEQ ID NO: 11)) derived from a modified KRAS polypeptide a molecule capable of binding may include, but is not limited to, an scFv sequence represented by any one of SEQ ID NO: 380, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, and SEQ ID NO: 393. For example, to bind to a modified peptide (e.g., ILDTAGHEEY (SEQ ID NO: 28), ILDTAGKEEY (SEQ ID NO: 30), ILDTAGLEEY (SEQ ID NO: 31), ILDTAGREEY (SEQ ID NO: 32)) derived from a modified H / K / N RAS polypeptide a molecule capable of binding may include, but is not limited to, an scFv sequence represented by any one of SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, and SEQ ID NO: 400. Examples of sequences (e.g., scFv sequences) capable of binding to specific modified peptides are shown in Table 3. In some cases, a molecule containing one or more antigen-binding domains (e.g., scFv) capable of binding to a modified peptide described herein (e.g., a modified peptide containing an amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) may have a sequence deviating from the sequences shown in Table 3 and may also be called a mutant sequence. For example one or more antigen-binding domains capable of binding to a modified peptide described herein (e.g., an scFv) may have a sequence deviating from the sequences shown in Table 3 and may also be called a mutant sequence. For example one or more antigen-binding domains capable of binding to a modified peptide described herein (e.g., an scFv) may have a sequence deviating from the sequences shown in Table 3 and may also be called a mutant sequence. For example one or more antigen-binding domains capable of binding to a modified peptide described herein (e.g., an scFv) may have a sequence deviating from the sequences shown in Table 3 and may also be called a mutant sequence. For example a molecule containing one or more antigen-binding domains (e.g., scFv) capable of binding to a modified peptide described herein may have a sequence deviating from the sequences shown in Table 3 and may also be called a mutant sequence. For example one or more antigen-binding domains capable of binding to a modified peptide described herein Molecules containing In can have at least 75% sequence identity (e.g., at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or more), provided that the mutant sequence maintains the ability to bind to the modified peptides described herein for any of the sequences shown in Table 3. Optionally, a molecule containing one or more antigen-binding domains capable of binding to the modified peptides described herein can contain any suitable set of the CDR sequences described herein, and any deviation from the sequences shown in Table 3 can be within the scaffold sequence(s). One property, at least 85% sequence identity, at least 90% sequence identity, at least 95 % sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least At least 98% sequence identity, at least 99% sequence identity, or more) is shown in Table 3 For any of the sequences, as long as the mutant sequence maintains the ability to bind to the modified peptides described herein, it can be possessed. In some cases, a molecule containing one or more antigen-binding domains capable of binding to the modified peptides described herein can contain any suitable set of the CDR Sequences, and any deviation from the sequences shown in Table 3 can be Within the scaffold sequence(s) (singular or plural). Can contain, and any deviation from the sequences shown in Table 3 is Can be within the scaffold sequence(s) (singular or plural).

[0040] Modified peptides described herein (e.g., amino acid sequences represented by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 2 2, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and A molecule containing one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptide represented by any one of SEQ ID NO: 32) can be bound (e.g., covalently or non-covalently) to a label (e.g., a detectable label). The detectable label can be any suitable label. In some cases, the label can be used to assist in detecting the presence or absence of one or more modified peptides described herein . For example, the labeled molecules described herein can be used in vitro to detect cancer cells (e.g., cancer cells expressing the modified peptides described herein) in a sample obtained from a mammal . The detectable label can be any suitable label. In some cases, the label can be used to assist in detecting the presence or absence of one or more modified peptides described herein In this specification. For example, the labeled molecules described herein can be used in vitro to detect cancer cells (e.g., cancer cells expressing the modified peptides described herein) in a sample obtained from a mammal . For example, the labeled molecules described herein can be used in vitro to detect cancer cells (e.g., cancer cells expressing the modified peptides described herein) in a sample obtained from a mammal (e.g., cancer cells expressing the modified peptides described herein) in a sample obtained from a mammal It can be output. In some cases, a label (e.g., a detectable label) can be used to assist in determining the position of one or more modified peptides described in this specification. For example, the labeled molecule described in this specification can be used in vivo to monitor anti-tumor therapy and / or detect mammalian cancer cells (e.g., cancer cells expressing the modified peptide described in this specification). Examples of labels that can be conjugated to the molecules described in this specification include, but are not limited to, radionuclides, chromophores, enzymes, and fluorescent molecules (e.g., green fluorescent protein).

[0041] Molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described in this specification (e.g., modified peptides containing the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) can be bound (e.g., covalently or non-covalently) to a therapeutic agent. The therapeutic agent can be any therapeutic agent. In some cases, the therapeutic agent can be an anti-cancer agent. Examples of therapeutic agents that can be conjugated to the molecules described in this specification include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, mertansine / emtansine (DM1), ​​​​​​​​​​​​​​​These include, but are not limited to, terrier toxins, and anti-cancer drugs such as gelonin.

[0042] This document provides modified peptides described herein (e.g., SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ No. 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:3 1, and a modified peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 32) 1 comprising one or more antigen-binding domains (e.g., scFv) capable of binding to Methods of using one or more of the molecules are also provided. For example, one or more modified peptides one or more antigen-binding domains capable of targeting (e.g., binding to) (e.g., scFv) to treat patients with or suffering from cancer. A mammal suspected of having cancer can be evaluated and / or a cancer (e.g. and treating a mammal having a cancer that expresses one or more modified peptides. In some cases, one or more molecules can be attached to the modified peptide. and using these to treat patients suffering from or having cancer. The present invention relates to a method for detecting the presence of one or more modified peptides in a sample obtained from a mammal suspected of having cancer. In some cases, one that can bind to the modified peptide can be detected. Alternatively, one or more molecules comprising multiple antigen-binding domains can be administered to a cancer (e.g., a modified peptide The present invention can be administered to a mammal having a cancer (cancer expressing a tyrosine kinase inhibitor) to treat the mammal. One or more antigen-binding domains capable of binding to the modified peptides described herein. Administration of one or more molecules, including, to a mammal (e.g., a human) having cancer may be effective in treating the mammal.

[0043] As described herein, any type of mammal can be evaluated and / or treated. Examples of mammals that can be evaluated and / or treated as described herein include primates (e.g., humans and non-human primates such as chimpanzees, baboons, or monkeys), dogs, cats, pigs, sheep, rabbits, mice, and rats, but are not limited thereto. In some cases, the mammal can be a human.

[0044] A mammal can be evaluated and / or treated for a suitable cancer. In some cases, the cancer can express one or more of the modified peptides described herein (e.g., one or more MANA). The cancer can be a primary cancer. The cancer can be a metastatic cancer. The cancer can include one or more solid tumors. The cancer can include one or more non-solid tumors. Examples of cancers that can be evaluated and / or treated as described herein (e.g., based at least in part on the presence of one or more of the modified peptides described herein) and / or treated as described herein (e.g., by administering one or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein) include blood cancers (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, leukemias such as acute myeloid leukemia (AML), and myeloma), lung cancer, pancreatic cancer, stomach cancer, colon cancer (e.g., colorectal cancer), ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, Cancer, bone and soft tissue cancer, breast cancer, prostate cancer, esophageal cancer, gastric cancer, kidney cancer, head and neck cancer, brain tumors (e.g., glioblastoma multiforme and astrocytoma) are included, but are not limited thereto.

[0045] When evaluating a mammal having cancer or suspected of having cancer, a modified peptide described herein (e.g., SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24 , SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, any one of the amino acid sequences represented by) can be used with one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptide, and the presence or absence of one or more modified peptides described herein can be evaluated. For example, the presence or absence, or level, of one or more modified peptides described herein in a sample obtained from a human can be used to determine whether the human has cancer. In some cases, the presence of one or more modified peptides described herein in a sample obtained from a mammal can be used to identify that the mammal has cancer. For example, a mammal can be identified as having cancer if a sample obtained from the mammal has one or more modified peptides described herein.

[0046] Any suitable sample obtained from a mammal can be evaluated for the presence or absence, or level, of one or more modified peptides described herein. For example, biological samples such as tissue samples (e.g., breast tissue) , and body fluid samples (e.g., blood, serum, plasma, or urine) can be obtained from a mammal obtained from, and can be evaluated for the presence or level of one or more modified peptides described herein using any suitable method, the presence or level of one or more modified peptides described herein can be detected. For example, Sanger sequencing single, chemical sequencing, nanopore sequencing, sequencing by ligation (SOLiD sequencing), sequencing using mass spectrometry, etc., but not limited to these sequencing techniques, the presence or level of one or more modified peptides described herein in a sample obtained from a mammal can be determined .

[0047] When treating a mammal having cancer, one or more antigen-binding domains (e.g., scFv) that can bind to a modified peptide described herein (e.g., an amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) can be administered to the mammal having cancer to treat the mammal. In some cases, the mammal may have cancer that expresses one or more modified peptides described herein. For example, one or more molecules containing one or more antigen-binding domains that can bind to the modified peptides described herein can be administered to a mammal having cancer that expresses the modified peptide One or more molecules capable of binding to AR and / or one or more scDb) and comprising one or more scFvs can be administered to a mammal having a cancer that expresses the modified peptide, thereby treating the mammal. In some cases, one or more molecules comprising one or more scFvs capable of binding to the modified peptides described herein (e.g., the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) can be administered to a mammal (e.g., a mammal having cancer) one or more times over a period of several days to several weeks. In some cases, one or more molecules comprising one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptides described herein can be formulated into a pharmaceutically acceptable composition for administration to a mammal. For example, the effective amount of one or more molecules comprising one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptides described herein can be formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. The pharmaceutical composition can be formulated for administration in solid or liquid form, including but not limited to sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions described herein include, but are not limited to, carriers well known in the art for formulating pharmaceutical compositions.

[0048] In some cases, one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptides described herein (e.g., the modified peptides containing the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) can be administered to a mammal (e.g., a mammal having cancer) one or more times over a period of several days to several weeks. In some cases, one or more molecules comprising one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptides described herein can be formulated into a pharmaceutically acceptable composition for administration to a mammal. For example, the effective amount of one or more molecules comprising one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptides described herein can be formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. The pharmaceutical composition can be formulated for administration in solid or liquid form, including but not limited to sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions described herein include, but are not limited to, carriers well known in the art for formulating pharmaceutical compositions. In some cases, one or more molecules comprising one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptides described herein can be administered to a mammal one or more times over a period of several days to several weeks. In some cases, one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptides described herein can be formulated into a pharmaceutically acceptable composition for administration to a mammal. For example, the effective amount of one or more molecules comprising one or more antigen-binding domains (e.g., scFv) capable of binding to the modified peptides described herein can be formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. The pharmaceutical composition can be formulated for administration in solid or liquid form, including but not limited to sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions described herein include, but are not limited to, carriers well known in the art for formulating pharmaceutical compositions. The pharmaceutical composition can be formulated for administration in solid or liquid form, including but not limited to sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions described herein include, but are not limited to, carriers well known in the art for formulating pharmaceutical compositions. The fillers and solvents include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin, but are not limited thereto.

[0049] One or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein (e.g., modified peptides containing the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) can be designed for oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or intratumoral administration. Compositions suitable for parenteral administration may include antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, including aqueous and non-aqueous sterile injection solutions. The formulations can be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and a sterile liquid carrier for injection, such as water, can be added immediately before use. ​​​It can be stored in a lyophilized (freeze-dried) state that only needs to be added. Sterile powders, granules, and tablets may be used to prepare immediate injection solutions and suspensions from the agents.

[0050] One or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein (e.g., modified peptides comprising the amino acid sequences set forth in any one of SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:2 2, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32) can be administered to any suitable location using any suitable technique. One or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein can be administered locally (e.g., intratumorally) or systemically. For example, the compositions provided herein can be administered locally by intratumoral administration (e.g., injection into the tumor) or administration into a biologically invaded space (e.g., intrathecal administration, intracerebellar administration, intraperitoneal administration and / or pleural administration). For example, the compositions provided herein can be administered systemically by oral administration to a mammal (e.g., a human) or intravenous administration (e.g., injection or infusion). The effective dose can vary depending on the risk and / or severity of cancer, the route of administration, the age and general health status of the subject, the use of excipients, the possibility of combination with other therapeutic treatments such as the use of other drugs, as well as the judgment of the treating physician. The modified peptides described herein (e.g., For example, the compositions provided herein can be administered systemically by oral administration to a mammal (e.g., a human) or intravenous administration (e.g., injection or infusion). or intravenous administration (e.g., injection or infusion) to a mammal (e.g., a human).

[0051] The effective dose can vary depending on the risk and / or severity of cancer, the route of administration, the age and general health status of the subject, the use of excipients, the possibility of combination with other therapeutic treatments such as the use of other drugs, as well as the judgment of the treating physician. The modified peptides described herein (e.g., then, one or more antigen-binding domains that can bind to a modified peptide comprising the amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) An effective amount of a composition comprising one or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to a modified peptide ( e.g., scFv) can be any amount that treats cancer present in a subject without causing significant toxicity in the subject. If a particular subject does not respond to a particular amount, the amount of one or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein can be increased (e.g., by 2-fold, 3-fold, 4-fold, or more). After administration of this higher amount, the mammal can be monitored for both responsiveness to treatment and toxic symptoms, and adjustments can be made accordingly. The effective amount can be maintained constant or adjusted as a sliding scale or variable dose depending on the subject's response to treatment. The actual effective amount used for a particular administration is affected by a variety of factors. For example, the frequency of administration, the duration of treatment, the use of multiple therapeutic agents, the route of administration, and the severity of the condition (e.g., cancer) may require an increase or decrease in the actual effective amount administered. The modified peptides described herein (e.g., SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 2 2, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) For example, the actual effective amount administered may need to be increased or decreased depending on the severity of cancer or other conditions.

[0052] The modified peptides described herein (e.g., SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 2 2, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and (a modified peptide comprising an amino acid sequence represented by any one of SEQ ID NO: 32) and bind thereto One or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind The dosing frequency of the one or more molecules can be any frequency that effectively treats a mammal having cancer without causing significant toxicity to the mammal. For example, the dosing frequency of one or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein can be from about 2 to about 3 times per week to about 2 to about 3 times per year. In some cases a subject having cancer can receive a single administration of one or more of the antibodies described herein. The dosing frequency of one or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein can be constant or can be changed during the course of treatment. The course of treatment with a composition comprising one or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein can include a drug-free period. For example a composition comprising one or more molecules comprising one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein can be administered once a month over a 2-year period, followed by a 6-month drug-free period, and such regimens can be repeated multiple times. Similar to the effective amount a variety of factors can affect the actual dosing frequency used for a particular administration. For example the effective amount, treatment duration, use of multiple therapeutic agents route of administration, and severity of the condition (e.g., cancer) may require an increase or decrease in the dosing frequency. One or more antigen-binding domains (e.g., scFv) that can bind to the modified peptide described herein The course of treatment with a composition comprising one or more molecules can include a drug-free period. For example One or more antigen-binding domains that can bind to the modified peptides described herein (e.g., scFv) can be administered once a month over a 2-year period, followed by a 6-month drug-free period, and such regimens can be repeated multiple times. Similar to the effective amount a variety of factors can affect the actual dosing frequency used for a particular administration. For example the effective amount, treatment duration, use of multiple therapeutic agents route of administration, and severity of the condition (e.g., cancer) may require an increase or decrease in the dosing frequency. The dosing frequency may need to be increased or decreased depending on the effective amount, treatment duration, use of multiple therapeutic agents route of administration, and severity of the condition (e.g., cancer).

[0053] One or more antigen-binding domains (e.g., scFv) that can bind to a modified peptide (e.g., a modified peptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 2 2, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) described herein) are included in one or more molecules. The effective period for administering the composition containing such one or more molecules can be any period that effectively treats cancer present in a mammal without causing significant toxicity to the mammal. In some cases, the effective period can vary from several months to several years. Generally, the effective period for treating a mammal with cancer can range from about 1 or 2 months to 5 years or more. The actual effective period used for a particular treatment can be affected by multiple factors. For example, the effective period can vary depending on the administration frequency, effective dose, use of multiple therapeutic agents, administration route, and severity of symptoms during treatment. In certain examples, it is possible to monitor cancer in a mammal to evaluate the effectiveness of cancer treatment. It is possible to determine whether to treat a mammal having cancer using any suitable method. For example, imaging techniques or laboratory assays can be used to evaluate the number of cancer cells and / or the size of tumors present in the mammal. For example, imaging techniques or laboratory assays can be used to evaluate the location of cancer cells and / or tumors present in the mammal.

[0054]

[0055] ​​​​​​​​​​In some cases, one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein (e.g., the amino acid sequences represented by any one of SEQ ID NO: 1, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) can be included in one or more molecules, which can be administered to a mammal having cancer as a combination therapy with one or more additional cancer treatments (e.g., anti-cancer agents). Cancer treatment can include any suitable cancer treatment. In some cases, cancer treatment can include surgery. In some cases , cancer treatment can include radiotherapy. In some cases, cancer treatment can include the administration of one or more therapeutic agents (e.g., one or more anti-cancer agents). Examples of anti-cancer agents include platinum compounds (e.g., cisplatin or carboplatin), taxanes (e.g., paclitaxel, docetaxel, or albumin-bound paclitaxel such as nab-paclitaxel), altretamine, capecitabine, cyclophosphamide, etoposide ( vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, luteinizing hormone-releasing hormone (LHRH) agonists (e.g., goserelin and leuprorelin ), anti-estrogens (e.g., tamoxifen), aromatase inhibitors (e.g., letrozole, anastrozole, exemestane), angiogenesis inhibitors (e.g., bevacizumab ), poly(ADP)-ribose polymerase (PARP) inhibitors (e.g., olaparib ), etc. ​ (such as olaparib, rucaparib, and niraparib), radioactive phosphorus, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, IL-2, and other cytokines, and any combination thereof, but not limited thereto. One or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein When one or more molecules containing are used in combination with one or more additional cancer therapies, the one or more additional cancer therapies can be administered simultaneously or independently. For example, a composition containing one or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein can be administered first, and then one or more additional cancer therapies can be administered, or vice versa. In addition, a kit containing one or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein (e.g., modified peptides containing the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) is also provided herein. For example, the kit can contain a composition (e.g., a pharmaceutically acceptable composition) containing one or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein. In some cases, the kit may contain instructions for performing any of the methods described herein. When one or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein are used in combination with one or more additional cancer therapies, the one or more additional cancer therapies can be administered simultaneously or independently. For example, a composition containing one or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein can be administered first, and then one or more additional cancer therapies can be administered, or vice versa.

[0056] In addition, a kit containing one or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein (e.g., modified peptides containing the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) is also provided herein. For example, the kit can contain a composition (e.g., a pharmaceutically acceptable composition) containing one or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein. In some cases, the kit may contain instructions for performing any of the methods described herein. In addition, a kit containing one or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein (e.g., modified peptides containing the amino acid sequences shown by any one of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32) is also provided herein. For example, the kit can contain a composition (e.g., a pharmaceutically acceptable composition) containing one or more molecules containing one or more antigen-binding domains (e.g., scFv) that can bind to the modified peptides described herein. In some cases, the kit may contain instructions for performing any of the methods described herein. In some cases, the kit may contain instructions for performing any of the methods described herein. Instructions for performing may be included. In some cases, the kit may include at least one dose of any of the compositions described herein (e.g., pharmaceutical compositions). In some cases, the ki tt may provide means (e.g., a syringe) for administering any of the compositions described herein (e.g., pharmaceutical compositions).

[0057] The present invention will be further illustrated by the following examples, which do not limit the scope of the present invention described in the claims.

Example

[0058] (Example 1: Identification of additional MANA body clones and conversion of MANA body clones to T cell-based therapeutic types) In this study, two phage display libraries were designed and constructed, both displaying single-chain variable fragments (scFv) on the phage surface. The scFv present in both libraries were based on a humanized 4D5 (trastuzumab) framework with introduced variability of amino acids at important positions in the complementarity-determining regions (CDRs) of the scFv.

[0059] Using the phage display library, scFv specifically recognizing mutant-containing peptides folded into complexes with recombinant HLA allele alpha chains and beta-2 microglobulin (b2M) were identified. These complexes, also referred to herein as monomers, mimic the natural peptide / HLA complexes on the cancer cell surface.

[0060] Peptide HLA targets may include the mutant peptides shown in Table 1 (e.g., mutation-related neoantigens (MAN A)). Phases that can specifically bind to the peptide HLA targets in Table 1 The complementary determining regions (CDRs) are shown in Table 2. Peptides that specifically bind to the HLA targets in Table 1 The scFvs that can do so are shown in Table 3. These scFvs can bind to mutation-related neoantigens and can also be called MANA bodies because of their ability to do so.

[0061]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

[0062]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

[0063] IDH2 peptide (SPNGTIQ NIL; SEQ ID NO: 1) containing the R140Q mutation in the complex with HLA-B7, and representative ELISA data of the scFv that specifically recognizes it are shown in Figure 1. The scFv is the wt version of the peptide of interest in the complex with the same HLA allele ​did not recognize. scFvs did not recognize other control peptides in the complex with HLA alleles when tested for binding to ELISA plates coated with monomers.

[0064] Furthermore, using flow cytometry, it was shown that scFv clones of the MANA body specifically stained HLA allele-matched cell lines when these cells were pulsed with mutant peptides rather than wt peptides or other control peptides (Figure 2-14).

[0065] To demonstrate that MANA body clones can be utilized as a therapy, selected MANA body clones were incorporated into CAR-T cells. Chimeric antigen receptor (CAR) T cells (CART) were engineered to recognize and kill cells expressing oncogenic mutation-containing peptides via the endogenous processing and presentation machinery of HLA molecules. Specifically, CARs targeting mutant KRAS G12V peptides presented in HLA-A3 and CARTs targeting mutant IDH2 R140Q peptides presented in HLA-B7 were engineered. MANA body scFvs targeting either mutant peptide were grafted into third-generation CAR constructs, and the CAR receptor was expressed in CD3+ T cells by mRNA electroporation. Subsequently, the CAR-T cells were co-cultured with COS-7 cells co-transfected with plasmids encoding KRAS / IDH2 mutants and wt proteins in combination with their respective HLA. T cells, including CAR-T cells, produce cytokines upon activation by allogeneic antigens on target cells, so the co-culture supernatant ​​​​​​​​​​​​​​​The release of IFNγ from the mutant and homologous H cells was measured by ELISA. IFNγ levels above background only when cotransfected with LA plasmid There was a significant release of β-heptane (β-heptane) (Figure 15). CAR-T cells co-cultured with S-7 cells released only background levels of IFNγ. Taken together, these findings support the efficacy and safety of CAR-T cells expressing the MANA Body clone. The cells can target tumor cells expressing MANA, which is presented on HLA molecules. This suggests that

[0066] To demonstrate that MANA body clones can be used as a therapeutic approach, we The ANA body clones were integrated into a bispecific antibody. One antibody binds to the target cancer cell. The antibody fragment binds to the CD3 protein on the surface of T cells. We designed a bispecific antibody that contains human CD3 epsilon, delta, and / or There are many different anti-CD3 scFv clones that target the gamma molecule. Examples of clones are shown in Table 4.

[0067] One antibody that binds to the mutant KRAS G12V peptide presented in HLA-A3 A single antibody fragment that has a cytoplasmic fragment and binds to the CD3 protein on the surface of T cells. Specifically, we designed a bispecific antibody bearing a variant presented in HLA-A3. We designed a bispecific antibody targeting a heterologous KRAS G12V peptide and CD3, and Mutant IDH2 R140Q peptide presented in A-B7 and CD3-targeting We designed a bispecific antibody that [Table 4-1]

Table 4-2

[0068] Three IDH2 R140Q combined with two different anti-CD3 scFv clones Representative scDb co-culture results of HLA-B7 MANA body scFv clones are shown in Figure 1 6A. T cells were co-transfected with COS-7 cells transfected with plasmids encoding HLA-B7, full-length IDH2 variants, and / or GFP and the indicated concentrations of scDb and co-cultured in the presence of. As a readout of T cell activation by the alloantigen on the target cells, the release of IFNγ in the co-culture supernatant was measured by ELISA. Only when COS-7 cells were co-transfected with HLA- B7 and mutant IDH2 R140Q plasmids was there significant T cell release of IFNγ above background, and the level of IFNγ was dependent on the concentration of scDb included in the well. T cells co-cultured with COS-7 cells co-transfected with HLA- B7 and wt IDH2 released only background levels of I FNγ. Representative scDb co-culture results of the KRAS G12V HLA-A3 MANA body scFv clone combined with an anti-CD3 clone into a single-chain diabody are shown in Figure 16B. In this co-culture, the single-chain diabody was tested at concentrations of 0, 50, and 1 00 ng / mL. Only when COS-7 cells were co-transfected with HLA-A3 and mutant KRAS G12V plasmids was there significant T cell release of IFNγ above background. T cells co-transfected with HLA-A3 and wt KRAS released only background levels of I FNγ. clone into a single-chain diabody are shown in Figure 16B. In this co-culture, the single-chain diabody was tested at concentrations of 0, 50, and 1 00 ng / mL. Only when COS-7 cells were co-transfected with HLA-A3 and mutant KRAS G12V plasmids was there significant T cell release of IFNγ above background. T cells co-transfected with HLA-A3 and wt KRAS released only background levels of I FNγ. T cells co-cultured with transfected COS-7 cells released IFNγ at background levels similar to those seen in wells without T cells, without target cells, and without scDb Only. Along with its isogene HLA-A3 knockout control, the endogenous KRAS G12V HLA-A3 positive cell line NCI-H441 as a target cell line was used. IFNγ release was only seen for the parental NCI-H441 cell line and not for HLA-A3 knockout NCI -H441. Together, these findings suggest bispecific antibodies containing MANA body clones targeting tumor cells expressing MANA presented in HLA molecules . To evaluate the effectiveness of using MANA body clones as a therapy, the CellTiter-Glo reagent from Promega was used to assay the viability of target cells of KRAS G12V HLA -A3 single-chain diabody (Figure 17). CellTiter-Glo measures the ATP concentration in the well, which is proportional to the number of live cells. By subtracting the CellTiter-Glo value from wells with T cells and normalizing to wells with only target cells, the viability of target cells was measured. Significant target cell death was only observed when NCI-H441 parental cells were incubated with T cells in the presence of KRAS G12V-A3 scDb or the pan-HLA-A3 scDb positive control. No target cell death was observed between the absence of scDb or in NCI-H441 HLA-A3 knockout wells

[0069] . G12V-A3 scDb or the pan-HLA-A3 scDb positive control. No target cell death was observed between the absence of scDb or in NCI-H441 HLA-A3 knockout wells . CellTiter-Glo measures the ATP concentration in the well, which is proportional to the number of live cells. By subtracting the CellTiter-Glo value from wells with T cells and normalizing to wells with only target cells, the viability of target cells was measured. NCI-H441 parental cells were incubated with T cells in the presence of KRAS G12V-A3 scDb or the pan-HLA-A3 scDb positive control. No target cell death was observed between the absence of scDb or in NCI-H441 HLA-A3 knockout wells . G12V-A3 scDb or the pan-HLA-A3 scDb positive control. No target cell death was observed between the absence of scDb or in NCI-H441 HLA-A3 knockout wells . No target cell death was observed between the absence of scDb or in NCI-H441 HLA-A3 knockout wells .

[0070] Together, these findings suggest redirecting T cells and Activate and kill tumor cells that express specific mutant proteins and HLA allele pairs (e.g., IDH2 R140Q and HLA-B7 and KRAS G12V and HLA-A3). This has been demonstrated.

[0071] (Materials and Methods) (Cells and Cell Lines) RPMI-6666 cells (ATCC, Manassas, Virginia) were cultured in RPMI-1640 (ATCC) containing 20% FBS( GE Hyclone, Logan, Utah, USA) and 1% penicillin-streptomycin (Life Technologies). T2 cells (ATCC) and MINO cells (ATCC) were cultured in RPMI-1640 (ATCC) containing 10% FBS(G E Hyclone) and 1% penicillin-streptomycin (Thermo Fisher ). T2A3 cells (gifted by Dr. Eric Lutz) were cultured in RPMI-1640 (ATCC) containing 10% FBS (GE Hyclone), 1% penicillin -streptomycin (Thermo Fisher), 0.1 mM MEM non-essential amino acids (NEAA, Thermo Fisher), and 500 μg / mL geneticin (T hermo Fisher). SigM5 cells (DSMZ, Braunschweig, Germany) were cultured in Iscove's MDM (ATCC) containing 20% FBS (GE Hy clone) and 1% penicillin-streptomycin (Thermo Fisher). Hs611.T cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (ATCC) containing 1 0% FBS (GE Hyclone) and 1% penicillin-streptomycin (Ther mo Fisher). N 0% FBS (GE Hyclone) and 1% penicillin-streptomycin (Ther mo Fisher). ​CI-H441 cells (ATCC) and COS-7 cells (ATCC) were cultured in McCoy's 5A (modified) medium (Thermo Fisher) containing 10% FBS ( GE Hyclone) and 1% penicillin-streptomycin (Thermo Fis her). COS-7 cells (ATCC, CRL-1651 (trademark)) were cultured in DMEM (high glucose, pyruvate; Thermo Fisher) containing 10% FBS (GE H yclone) and 1% penicillin-streptomycin (Thermo Fisher and). 293FT cells (Thermo Fisher) were cultured in high glucose D-MEM (Thermo Fisher containing 10% FBS (GE Hyc lone), 0.1 mM MEM non-essential amino acids (NEAA, Thermo Fish er), 6 mM L-glutamine (Thermo Fisher), 1 mM MEM sodium pyruvate (Thermo Fisher), 500 μg / ml genetici n (Thermo Fisher), and 1% penicillin-streptomycin (The rmo Fisher). All cell lines were maintained at 37 °C under 5% CO2. PBMCs were obtained by Ficoll-Paque PL US (GE Healthcare) gradient centrifugation of whole blood from healthy volunteer donors. CD3+ cells were positively selected from PBMCs using CD

[0072] 3 microbeads (Miltenyi Biotec) and activated and expanded with Dynabeads® Human T-Activator CD3 / CD28 (Life Technologies). Spec ifically activated and proliferated with Dynabeads® Human T-Activator CD3 / CD28 (Life Technologies). ​Unless otherwise specified, naïve CD3+ T cells were cultured in RPMI-1640 (ATCC) containing 10% FBS (GE Hyclone), 1% penicillin-streptomycin (Life Technologies), and 10 0 IU / mL recombinant human interleukin-2 (Proleukin®) at 37 °C under 5% CO2.

[0073] (Construction of phage display libraries) For the first-generation phage library, oligonucleotides were synthesized by DNA 2.0 (Menlo Park, CA) using mixed and split-pool degenerate oligonucleotide synthesis. For the second-generation phage library, oligonucleotides were synthesized using trinucleotide mutagenesis (TRIM) technology by GeneArt® (Thermo Fisher, Gaithersburg, MD). For both libraries, the oligonucleotides were incorporated into the pADL-10b phagemid (Antibody Design Labs, San Diego, CA). This phagemid contains an F1 origin of replication, a transcriptional repressor that restricts non-induced expression, a lac operator -promoter, and a lac repressor. The scFv was synthesized using the pelB periplasmic secretion signal peptide and subcloned downstream of the lac operator. In the first-generation library, a myc epitope tag followed by a TEV protease cleavage recognition sequence was placed immediately downstream of the variable heavy chain, and in the second-generation library, the scFv was followed by a FLAG tag. Following the scFv, tag, and cleavage site, there was a full-length in-frame M13 pIII coat protein sequence.

[0074] ​​​​​​​ To transform phagemid DNA into bacteria, 10–20 ng of ligation product was used. Place the material on ice and incubate with 10 μL of electrocompetent SS320 cells (Lucigen, Mido The mixture was mixed with 14 μL of GeCl4 (100 μL, St. Louis, MO) and 14 μL of double distilled water. ne Pulser electroporation system (Bio-Rad, Hercules Electroporation was performed using Recovery Med (RME, CA) and The ligation product was recovered in 60 ng of ia (Lucigen) at 37°C for 60 min. The transformed cells were pooled and cultured in carbenicillin (100 μg / mL) and 2% glucose. The cells were seeded on 24cm x 24cm plates containing supplemented 2xYT medium. The cells were incubated at 37°C for 6 h. The cells were grown for 2 h and then placed at 4° C. overnight. Transformation efficiency for each series of electroporation To determine the potency of the antibody, an aliquot was removed and titered by serial dilution. The grown cells were incubated in 850 ml of 100 mL of 100% glucose-containing carbenicillin (100 μg / mL). 2xYT medium to a final OD600 of 5-15. Take 1 mL of the culture and dilute approximately 1:200 to achieve a final OD600 of 0.05–0.07. The remaining cultures were added with 150 mL of sterile glycerol before being flash frozen. A diluted bacteria was grown until the OD600 reached 0.2-0.4. M13K07 helper phage (Antibody Design Labs, San The cultures were infected with 1000 ng / ml sera from the San Diego, CA, USA and shaken at 37°C for 1 hour. The cells were then incubated with carbenicillin (100 μg / mL) and kanamycin (50 μg / mL). Resuspended in 2xYT medium containing and grown overnight at 30°C for phage production. The next morning, the bacterial culture was aliquoted into 50 mL Falcon tubes and pelleted twice at high speed to clarify the supernatant. The supernatant containing phage was precipitated on ice for 40 minutes using a 20% PEG-8000 / 2.5 M NaCl solution at a ratio of 4:1 PEG / NaCl to supernatant. After precipitation, the phage was centrifuged at 12,000 g for 40 minutes and resuspended in 1 mL vol 1X TBS, 2 m M EDTA. Phages from multiple tubes were pooled, reprecipitated, and resuspended to an average titer of 1 x10 13 cfu / mL. In the first-generation library, the total number of transformants obtained was 5.5x10 . In the second-generation library, the total number of transformants obtained was 3.6x10 9 . Each library was aliquoted and stored at -80°C in 15% glycerol . 10

[0075] (Next-generation sequencing of the complete phage library) DNA from the library was amplified using primers adjacent to the CDR-H3 region. Molecular barcodes are incorporated into the sequences at the 5´ ends of these primers, facilitating unambiguous enumeration of individual phage sequences. The protocols for PCR amplification and sequencing are described by Kinde et al. Sequences were processed and translated using a custom SQL database, and both nucleotide sequences and amino acid translations were analyzed using Microsoft Excel.

[0076] (Peptides and HLA monomers) Mutants, wt, and control peptides (listed in Table 1) were analyzed using NetMHC version 4.0 ​​​​was predicted to bind to HLA alleles. All peptides were synthesized by >90% purity by 0 (CHANTILLY, VA). Peptides were resuspended in DM SO or DMF at 10 mg / mL and stored at -20 °C. HLA monomers were synthesized by refolding recombinant HLA with peptides and beta2-microglobulin, purified by gel filtration, and biotinylated (Fred Hutchins on Immune Monitoring Lab, Seattle, WA). ELI SA was performed using the W6 / 32 antibody (BioLegend, San Diego, CA) to confirm that the monomers were folded prior to selection .

[0077] (Selection of phage bound to mutant peptide-HLA monomers) Biotinylated monomers containing HLA and beta-2-microglobulin proteins were bound to MyOne T1 streptavidin magnetic beads (Life Technologies , Carlsbad, CA). Biotinylated monomers were incubated with 30 μL of MyOne T1 beads (per 1 μg of monomer) in blocking buffer (PBS, 0.5% BSA, 0.1% Na-azide) for 1 hour at room temperature (RT). After the first incubation, the complex was washed 3 times with 1 mL of blocking buffer and resuspended in 1 mL of blocking buffer.

[0078] (Enrichment step) In the first enrichment step of the selection, phage representing a 1000-fold coverage of the library were added to bare washed MyOne T1 beads and heat-denatured bead-bound HLA mono Incubated with the marker on the rotor at 4 °C overnight. This step was necessary to remove any phage that recognized streptavidin or denatured monomers that were present in trace amounts in all preparations of the biotinylated monomer After negative selection, the beads were separated with a DynaMag-2 magnet (Life Technologies), and the supernatant containing unbound phage was transferred for positive selection against 1 μg of mutant peptide HLA monomer bound to MyOne T1 streptavidin magnetic beads Before elution, the beads were washed 10 times with 1 mL of 1X TBS containing 0.5% Tween-20 using a magnet The phage was eluted by resuspending the beads in 1 mL of 0.2 M glycine, pH 2.2. After a 10-minute incubation, 150 μL of 1 M Tris, pH 9.0 was added to neutralize the solution The neutralized phage was used to infect a 10 mL culture of mid-log phase SS320, and M13K07 helper phage (MOI of 4) and 2% glucose were added. After shaking at 37 °C for 1 hour, the bacteria were resuspended in 2xYT medium containing carbenicillin (100 μg / mL), kanamycin (50 μg / mL), and 50 μM IPTG, and cultured overnight at 30 °C for phage production The next morning, the phage was precipitated with PEG / NaCl as described above

[0079] (Final selection step) Using the phage generated in the enrichment step, 3 to 5 final selections were performed. For each round of final selection, 10 - 0.1% of the precipitated phage was used against HLA allele-matched cells lacking the target mutant protein for the first negative selection. Then, the unbound pha ​​​​​​​​​​​The library was negatively selected against the native wt peptide-HLA monomer and irrelevant HLA-allele-matched monomers. After negative selection, the beads were separated with a DynaMag 2 magnet (Life Technologies) and the supernatant containing unbound phage was transferred for positive selection with 250 ng - 1 μg of mutant peptide-HLA monomer as described in the enrichment phase. (ELISA) Streptavidin-coated 96-well plates (R&D Systems, Minneapolis, MN) were coated overnight at 4°C with 50 ng (in 50 μL) of biotinylated mutant or wt peptide-HLA monomer in blocking buffer (PBS containing 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide). The plates were briefly washed with 1X TBST (TBS + 0.05% Triton-X 100). Phage were serially diluted to the indicated concentrations in blocking buffer and 50 μL was added to each well. After incubating the phage for 2 hours at RT, the plates were washed (ELISA plate washer (BioTek, Winooski, VT) was used to wash 6 times with 1X TBS-0.05% Tween-20 (TBST)). Bound phage were diluted 1:3000 in 1X TBST and incubated for 1 hour at room temperature with 50 μL of rabbit anti-M13 antibody (Pierce, Rockford, IL), then washed 6 more times and incubated for 1 hour at room temperature with 50 μL of anti-rabbit HRP (Thermo Fisher) diluted 1:10,000 in 1X TBST. After the last 6 washes with 1X TBST, 50

[0080] (ELISA) Streptavidin-coated 96-well plates (R&D Systems, Minneapolis, MN) were coated overnight at 4°C with 50 ng (in 50 μL) of biotinylated mutant or wt peptide-HLA monomer in blocking buffer (PBS containing 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide). The plates were briefly washed with 1X TBST (TBS + 0.05% Triton-X 100). Phage were serially diluted to the indicated concentrations in blocking buffer and 50 μL was added to each well. After incubating the phage for 2 hours at RT, the plates were washed (ELISA plate washer (BioTek, Winooski, VT) was used to wash 6 times with 1X TBS-0.05% Tween-20 (TBST)). Bound phage were diluted 1:3000 in 1X TBST and incubated for 1 hour at room temperature with 50 μL of rabbit anti-M13 antibody (Pierce, Rockford, IL), then washed 6 more times and incubated for 1 hour at room temperature with 50 μL of anti-rabbit HRP (Thermo Fisher) diluted 1:10,000 in 1X TBST. After the last 6 washes with 1X TBST, 50 Streptavidin-coated 96-well plates (R&D Systems, Minneapolis, MN) were coated overnight at 4°C with 50 ng (in 50 μL) of biotinylated mutant or wt peptide-HLA monomer in blocking buffer (PBS containing 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide). The plates were briefly washed with 1X TBST (TBS + 0.05% Triton-X 100). Phage were serially diluted to the indicated concentrations in blocking buffer and 50 μL was added to each well. After incubating the phage for 2 hours at RT, the plates were washed (ELISA plate washer (BioTek, Winooski, VT) was used to wash 6 times with 1X TBS-0.05% Tween-20 (TBST)). Bound phage were diluted 1:3000 in 1X TBST and incubated for 1 hour at room temperature with 50 μL of rabbit anti-M13 antibody (Pierce, Rockford, IL), then washed 6 more times and incubated for 1 hour at room temperature with 50 μL of anti-rabbit HRP (Thermo Fisher) diluted 1:10,000 in 1X TBST. After the last 6 washes with 1X TBST, 50 ​​​μL of TMB substrate (BioLegend, San Diego, CA) was added to the wells and the reaction was quenched with 1N sulfuric acid. Absorbance at 450 nm was measured with a Synergy H1 multi mode reader (BioTek, Winooski, VT).

[0081] Individual colonies of SS320 cells transformed with limiting dilutions of phage obtained from the final selection were selected to perform monoclonal phage ELISA. Individual colonies were inoculated into 200 μl of 2xYT medium containing 100 μg / ml carbenicillin and 2% glucose and grown at 37 °C for 3 hours. Next, the cells were infected with 1.6 x 10 7 M13K07 he lper phage (Antibody Design Labs, San Diego, CA ifornia) and incubated with shaking at 37 °C. The cells were pelleted and resuspended in 300 μl of 2xYT medium containing carbenicillin (100 μg / ml), kanamycin (50 μg / ml), and 50 u M IPTG and grown overnight at 30 °C. The cells were pelleted and the supernatant containing phage was used for ELISA as described above.

[0082] (Peptide Pulse and Flow Cytometry) For peptide pulse, HLA-matched cells were washed once with PBS and once with serum-free RPMI-164 0, then 10 6 cells per ml were incubated overnight at 37 °C in serum-free RPMI-1640 containing 50 μg / ml peptide and 10 μg / ml human beta2 microglobulin (ProSpec, East Brunswick, NJ ersey). The pulsed cells were pelleted and cold staining buffer (0.5% BSA, 2 mM EDTA, and ) was added to the pellet cells washed once with PBS containing 0.1% sodium azide and resuspended in 100 μL of staining buffer Phage staining was performed for 1 hour with 10 μL (approx. 1 x 10 9 ) of phage at a total volume of 10 0 μL on ice, followed by washing once with 4 mL of cold staining buffer. Next, the cells were stained with 1 μL of rabbit anti-M13 antibody (Pierce, Rockford, IL) at a total volume of 10 0 μL on ice for 1 hour and washed once with 4 mL of cold staining buffer. The cells were stained with anti rabbit-PE (Biolegend) at a total volume of 100 μL on ice for 1 hour and incubated with LIVE / DEAD Fixable Near-I R Dead Cell Stain (Thermo Fisher) for 10 minutes at room temperature according to the manufacturer's instructions. The cells were washed once with 4 mL of staining buffer and then resuspended in 300 μL of staining buffer before analysis The stained cells were analyzed using an LSRII flow cytometer (Becton Dickinson, Marlborough, MA achusetts).

[0083] (Construction and Generation of CAR) A third-generation chimeric antigen receptor (CAR) construct containing the MANA body scFv, CD28 transmembrane domain, and 4-1BB and CD3 ζ intracellular domains was synthesized (Gene Art®) and cloned into the mammalian expression vector pCI (Promega). mRNA was synthesized using the T7 mScript™ Standard mRNA Pro duction System Kit (CellScript®) according to the manufacturer's instructions. CAR mRNA was electroporated into cells using a BTX ECM 2001 Elec The tro Cell Manipulator (Harvard Apparatus) was used to electroporate primary CD3+ T cells to generate CAR-T cells.

[0084] (CAR-T activation co-culture assay) COS-7 cells were transfected in 96-well plate format with various combinations of pcDNA3. 1 (Life Technologies) plasmids encoding HLA-A3, HLA-B7, IDH2 (WT), IDH2 (R1 40Q), KRAS (WT), and KRAS (G12V) using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions. 100,000 electroporated CAR-T cells were overlaid on the transfected COS-7 cells, and the co-culture was incubated at 37 °C under 5% CO2 for 4 hours. After co-culture, the conditioned medium was collected and assayed for secreted IFNγ by ELISA (Quantikine®, R&D Systems).

[0085] (Bispecific antibody production) gBlocks encoding bispecific antibodies were ordered from IDT (Skokie, IL). According to the manufacturer's protocol, the gBlocks were topologically cloned into pcDNA3.4 plasmids (Thermo Fisher). 293FT cells (Thermo Fisher) were transfected with the bispecific antibody pcDNA3.4 plasmids in T75 flasks using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions. After 5 - 7 days of incubation, The medium was collected and centrifuged at 3,000 g for 10 minutes at 4°C. According to the manufacturer's instructions, Clontech Capturem (trademark) His-Tagged Purifica tion Miniprep Kit (Takara, Mountain View, Califor nia) was used to purify the bispecific antibody protein. According to the manufacturer's instructions, Z eba spin 7k MWCO desalting column was used to desalt the bispecific antibody protein into PBS. Mini-PROTEAN (registered trademark) TGX Stain-Fre e (trademark) Precast Gels (Biorad, Hercules, California) was used to quantify the bispecific antibody concentration using a standard curve of proteins of known concentration and a ChemiDoc XRS+ Imager (Biorad) was used to image the unstained gel.

[0086] (Bispecific antibody co-culture assay) COS-7 cells were transfected with various combinations of pcDNA3. 1 (Life Technologies) plasmids encoding HLA-A3, HLA-B7, IDH2 (wt), IDH2 (R1 40Q), KRAS (wt), and KRAS (G12V) according to the manufacturer's instructions using Lipofectamine3000 (Life Te chnologies) in a T75 flask. 50,000 T cells were combined with 30,000 transfected COS-7 cells or 10,000 NCI- H441 cells and the indicated concentration of bispecific antibody in a 96-well plate and co-cultured for 24 hours at 37°C under 5% CO2. After co-culture, the 96-well plate The cells were rapidly frozen, the conditioned medium lysate was recovered, and assayed for secreted IFNγ by ELISA (Quantikine® (registered trademark), R&D Systems). Alternatively, after co-culture, the viability of the target cells was measured using CellTiter-Glo (Promega).

[0087] (Other embodiments) The present invention has been described in conjunction with its detailed description, but it is to be understood that the foregoing description is exemplary, not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A molecule comprising an antigen-binding domain capable of binding to a peptide-HLA-beta-2 microglobulin complex, wherein the peptide comprises a modified peptide, the HLA is class I HLA, and the antigen-binding domain does not bind to a complex comprising the wild-type version of the modified peptide, the modified peptide is derived from a modified KRAS polypeptide, the modified peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 24, the antigen-binding domain comprises CDR-VL1 shown below, CDR-VL2 shown below, CDR-VL3 shown below, CDR-VH1 shown below, CDR-VH2 shown below, and CDR-VH3 shown below: CDR-VL1: SEQ ID NO: 33; CDR-VL2: SAS or SAY; CDR-VL3: SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 188, SEQ ID NO: 85, SEQ ID NO: 189, SEQ ID NO: 168, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, or SEQ ID NO: 274; CDR-VH1: SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, or SEQ ID NO: 402; CDR-VH2: SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, or SEQ ID NO: 282; and CDR-VH3: SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, or SEQ ID NO: 287 Molecule. **Claim 2**: The modified peptide contains the amino acid sequence of SEQ ID NO: 11, the class I HLA is HLA-B7, The molecule according to claim 1, wherein the antigen-binding domain contains any one of the following. (i) The CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 270, the CDR-VH1 is SEQ ID NO: 275, the CDR-VH2 is SEQ ID NO: 278, and the CDR-VH3 is SEQ ID NO: 283; (ii) The CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 271, the CDR-VH1 is SEQ ID NO: 402, the CDR-VH2 is SEQ ID NO: 279, and the CDR-VH3 is SEQ ID NO: 284; (iii) The CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 272, the CDR-VH1 is SEQ ID NO: 276, the CDR-VH2 is SEQ ID NO: 280, and the CDR-VH3 is SEQ ID NO: 285; (iv) The CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 273, the CDR-VH1 is SEQ ID NO: 277, the CDR-VH2 is SEQ ID NO: 281, and the CDR-VH3 is SEQ ID NO: 286; and (v) The CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 274, the CDR-VH1 is SEQ ID NO: 157, the CDR-VH2 is SEQ ID NO: 282, and the CDR-VH3 is SEQ ID NO: 287 **Claim 3**: The modified peptide comprises the amino acid sequence of SEQ ID NO: 18, the class I HLA is HLA-A2, the antigen-binding domain comprises any one of the following, the molecule according to claim 1. (i) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 110, the CDR-VH1 is SEQ ID NO: 112, the CDR-VH2 is SEQ ID NO: 115, the CDR-VH3 is SEQ ID NO: 118; (ii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 111, the CDR-VH1 is SEQ ID NO: 113, the CDR-VH2 is SEQ ID NO: 116, the CDR-VH3 is SEQ ID NO: 119; and (iii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 110, the CDR-VH1 is SEQ ID NO: 114, the CDR-VH2 is SEQ ID NO: 117, the CDR-VH3 is SEQ ID NO: 120 **Claim 4**: The modified peptide comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 22, the class I HLA is HLA-A3, the antigen-binding domain comprises the following, the molecule according to claim 1. (i) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 121, the CDR-VH1 is SEQ ID NO: 124, the CDR-VH2 is SEQ ID NO: 127, the CDR-VH3 is SEQ ID NO: 129 **Claim 5**: The modified peptide comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, the class I HLA is HLA-A3, the antigen-binding domain comprises any one of the following, the molecule according to claim 1. (i) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 122, the CDR-VH1 is SEQ ID NO: 125, the CDR-VH2 is SEQ ID NO: 128, the CDR-VH3 is SEQ ID NO: 130; (ii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 123, the CDR-VH1 is SEQ ID NO: 126, the CDR-VH2 is SEQ ID NO: 128, the CDR-VH3 is SEQ ID NO: 131 **Claim 6**: The modified peptide comprises the amino acid sequence of SEQ ID NO: 22, the class I HLA is HLA-A3, The molecule according to claim 1, wherein the antigen-binding domain comprises any one of the following: (i) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 132, the CDR-VH1 is SEQ ID NO: 137, the CDR-VH2 is SEQ ID NO: 142, and the CDR-VH3 is SEQ ID NO: 147; (ii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 133, the CDR-VH1 is SEQ ID NO: 138, the CDR-VH2 is SEQ ID NO: 143, and the CDR-VH3 is SEQ ID NO: 148; (iii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 134, the CDR-VH1 is SEQ ID NO: 139, the CDR-VH2 is SEQ ID NO: 144, and the CDR-VH3 is SEQ ID NO: 149; (iv) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 135, the CDR-VH1 is SEQ ID NO: 140, the CDR-VH2 is SEQ ID NO: 145, and the CDR-VH3 is SEQ ID NO: 150; and (v) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 136, the CDR-VH1 is SEQ ID NO: 141, the CDR-VH2 is SEQ ID NO: 146, and the CDR-VH3 is SEQ ID NO: 151

7. The modified peptide comprises the amino acid sequence of SEQ ID NO: 21, the class I HLA is HLA-A3, The molecule according to claim 1, wherein the antigen-binding domain comprises any one of the following: (i) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 152, the CDR-VH1 is SEQ ID NO: 156, the CDR-VH2 is SEQ ID NO: 160, and the CDR-VH3 is SEQ ID NO: 164; (ii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 153, the CDR-VH1 is SEQ ID NO: 157, the CDR-VH2 is SEQ ID NO: 161, and the CDR-VH3 is SEQ ID NO: 165; (iii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 154, the CDR-VH1 is SEQ ID NO: 158, the CDR-VH2 is SEQ ID NO: 162, and the CDR-VH3 is SEQ ID NO: 166; and (iv) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 155, the CDR-VH1 is SEQ ID NO: 159, the CDR-VH2 is SEQ ID NO: 163, and the CDR-VH3 is SEQ ID NO: 167

8. The modified peptide contains the amino acid sequence of SEQ ID NO: 21, the class I HLA is HLA-A11, The molecule according to claim 1, wherein the antigen-binding domain contains any one of the following. (i) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 168, the CDR-VH1 is SEQ ID NO: 173, the CDR-VH2 is SEQ ID NO: 178, and the CDR-VH3 is SEQ ID NO: 183; (ii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 169, the CDR-VH1 is SEQ ID NO: 174, the CDR-VH2 is SEQ ID NO: 179, and the CDR-VH3 is SEQ ID NO: 184; (iii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 170, the CDR-VH1 is SEQ ID NO: 175, the CDR-VH2 is SEQ ID NO: 180, and the CDR-VH3 is SEQ ID NO: 185; (iv) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 171, the CDR-VH1 is SEQ ID NO: 176, the CDR-VH2 is SEQ ID NO: 181, and the CDR-VH3 is SEQ ID NO: 186; and (v) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 172, the CDR-VH1 is SEQ ID NO: 177, the CDR-VH2 is SEQ ID NO: 182, and the CDR-VH3 is SEQ ID NO: 187

9. The modified peptide contains the amino acid sequence of SEQ ID NO: 24, the class I HLA is HLA-A11, The molecule according to claim 1, wherein the antigen-binding domain contains any one of the following. (i) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 188, the CDR-VH1 is SEQ ID NO: 196, the CDR-VH2 is SEQ ID NO: 207, and the CDR-VH3 is SEQ ID NO: 218; (ii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 85, the CDR-VH1 is SEQ ID NO: 197, the CDR-VH2 is SEQ ID NO: 208, and the CDR-VH3 is SEQ ID NO: 219; (iii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 189, the CDR-VH1 is SEQ ID NO: 198, the CDR-VH2 is SEQ ID NO: 209, and the CDR-VH3 is SEQ ID NO: 220; (iv) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 168, the CDR-VH1 is SEQ ID NO: 199, the CDR-VH2 is SEQ ID NO: 210, and the CDR-VH3 is SEQ ID NO: 221; (v) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 190, the CDR-VH1 is SEQ ID NO: 200, the CDR-VH2 is SEQ ID NO: 211, and the CDR-VH3 is SEQ ID NO: 222; (vi) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 191, the CDR-VH1 is SEQ ID NO: 201, the CDR-VH2 is SEQ ID NO: 212, and the CDR-VH3 is SEQ ID NO: 223; (vii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 192, the CDR-VH1 is SEQ ID NO: 202, the CDR-VH2 is SEQ ID NO: 213, and the CDR-VH3 is SEQ ID NO: 224; (viii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 191, the CDR-VH1 is SEQ ID NO: 203, the CDR-VH2 is SEQ ID NO: 214, and the CDR-VH3 is SEQ ID NO: 225; (ix) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 193, the CDR-VH1 is SEQ ID NO: 204, the CDR-VH2 is SEQ ID NO: 215, and the CDR-VH3 is SEQ ID NO: 226; (x) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 194, the CDR-VH1 is SEQ ID NO: 205, the CDR-VH2 is SEQ ID NO: 216, the CDR-VH3 is SEQ ID NO: 227; and (xi) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 195, the CDR-VH1 is SEQ ID NO: 206, the CDR-VH2 is SEQ ID NO: 217, and the CDR-VH3 is SEQ ID NO: 228

10. The modified peptide contains the amino acid sequence of SEQ ID NO: 22, the class I HLA is HLA-A11, The antigen-binding domain of the molecule according to claim 1 contains any one of the following. (i) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 229, the CDR-VH1 is SEQ ID NO: 234, the CDR-VH2 is SEQ ID NO: 240, and the CDR-VH3 is SEQ ID NO: 246; (ii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 230, the CDR-VH1 is SEQ ID NO: 235, the CDR-VH2 is SEQ ID NO: 241, and the CDR-VH3 is SEQ ID NO: 247; (iii) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 231, the CDR-VH1 is SEQ ID NO: 236, the CDR-VH2 is SEQ ID NO: 242, and the CDR-VH3 is SEQ ID NO: 248; (iv) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 232, the CDR-VH1 is SEQ ID NO: 237, the CDR-VH2 is SEQ ID NO: 243, and the CDR-VH3 is SEQ ID NO: 249; (v) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 168, the CDR-VH1 is SEQ ID NO: 238, the CDR-VH2 is SEQ ID NO: 244, and the CDR-VH3 is SEQ ID NO: 250; and (vi) the CDR-VL1 is SEQ ID NO: 33, the CDR-VL2 is SAS, the CDR-VL3 is SEQ ID NO: 233, the CDR-VH1 is SEQ ID NO: 239, the CDR-VH2 is SEQ ID NO: 245, and the CDR-VH3 is SEQ ID NO: 251

11. The molecule according to any one of claims 1 to 10, wherein the modified peptide contains 10 amino acids.

12. The modified peptide contains SEQ ID NO: 11, the class I HLA is HLA-B7, and the antigen-binding domain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 380, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, and SEQ ID NO:

393. The molecule according to claim 1 or 2.

13. The modified peptide contains SEQ ID NO: 18, the class I HLA is HLA-A2, and the antigen-binding domain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 338, SEQ ID NO: 339, and SEQ ID NO:

340. The molecule according to claim 1 or 3.

14. The modified peptide contains SEQ ID NO: 20 or SEQ ID NO: 22, the class I HLA is HLA-A3, and the antigen-binding domain contains the amino acid sequence of SEQ ID NO:

341. The molecule according to claim 1 or 4.

15. The modified peptide contains the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, the class I HLA is HLA-A3, and the antigen-binding domain contains the amino acid sequence of SEQ ID NO: 342 or SEQ ID NO:

343. The molecule according to claim 1 or 5.

16. The modified peptide contains the amino acid sequence of SEQ ID NO: 21, the class I HLA is HLA-A3, and the antigen-binding domain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, and SEQ ID NO:

352. The molecule according to claim 1 or 7.

17. The modified peptide contains the amino acid sequence of SEQ ID NO: 22, the class I HLA is HLA-A3, and the antigen-binding domain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, and SEQ ID NO:

348. The molecule according to claim 1 or 6.

18. The modified peptide contains the amino acid sequence of SEQ ID NO: 24, the class I HLA is HLA-A11, and the antigen-binding domain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, and SEQ ID NO:

368. The molecule according to claim 1 or 9. **Claim 19**: The molecule according to claim 1 or 8, wherein the modified peptide comprises the amino acid sequence of SEQ ID NO: 21, the class I HLA is HLA-A11, and the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, and SEQ ID NO:

357. **Claim 20**: The molecule according to claim 1 or 10, wherein the modified peptide comprises the amino acid sequence of SEQ ID NO: 22, the class I HLA is HLA-A11, and the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373, and SEQ ID NO:

374. **Claim 21** The molecule according to any one of claims 1 to 20, wherein the molecule is selected from the group consisting of an antibody, an antibody fragment, a single-chain variable fragment (scFv), a chimeric antigen receptor (CAR), a T cell receptor (TCR), a tandem scFv, a bispecific T cell engager, a diabody, a single-chain diabody, an scFv-Fc, a bispecific antibody, and a dual-affinity retargeting antibody (DART). **Claim 22** The molecule according to any one of claims 1 to 21, wherein the molecule is a single-chain diabody. **Claim 23** The molecule according to any one of claims 1 to 22, further comprising a second antigen-binding domain capable of binding to an effector cell receptor selected from the group consisting of CD3, CD28, CD4, CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, and CD27. **Claim 24**: The molecule according to claim 23, wherein the second antigen-binding domain is capable of binding to CD3, and the second antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 404, SEQ ID NO: 405, SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 411, SEQ ID NO: 412, SEQ ID NO: 413, SEQ ID NO: 414, SEQ ID NO: 415, SEQ ID NO: 416, and SEQ ID NO:

417. **Claim 25**: The molecule according to claim 23, wherein the second antigen-binding domain is capable of binding to CD16a. **Claim 26**: The molecule according to claim 23, wherein the second antigen-binding domain is capable of binding to NKG2D. **Claim 27** A chimeric antigen receptor (CAR) comprising: an extracellular domain comprising the antigen-binding domain according to any one of claims 1 to 26; a transmembrane domain; an intracellular domain and a CAR comprising the same.

28. The CAR according to claim 27, wherein the transmembrane domain comprises the transmembrane domain of CD4, CD8, or CD28.

29. The CAR according to claim 27 or claim 28, wherein the intracellular domain comprises one or more co-stimulatory domains from CD28, DAP10, ICOS, OX40, and / or 4-1BB.

30. The CAR according to any one of claims 27 to 29, wherein the intracellular domain comprises a signaling domain from CD3-zeta.

31. A T cell expressing the CAR according to any one of claims 27 to 30.

32. A pharmaceutical composition for treating a mammal having cancer, comprising: a molecule according to any one of claims 1 to 26, wherein the cancer comprises cancer cells expressing the modified peptide.

33. A pharmaceutical composition for treating a mammal having cancer, comprising: a T cell expressing the CAR according to any one of claims 27 to 30, wherein the cancer comprises cancer cells expressing the modified peptide.

34. The pharmaceutical composition according to claim 32 or 33, wherein the mammal is a human.

35. The pharmaceutical composition according to any one of claims 32 to 34, wherein the cancer is selected from the group consisting of Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, multiple myeloma, breast cancer, prostate cancer, esophageal cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, and astrocytoma.

36. Use of a molecule according to any one of claims 1 to 26 in the manufacture of a pharmaceutical for treating a mammal having cancer, wherein the cancer comprises cancer cells expressing the modified peptide.

37. Use of a T cell expressing the CAR according to any one of claims 27 to 30 in the manufacture of a pharmaceutical for treating a mammal having cancer, wherein the cancer comprises cancer cells expressing the modified peptide.

38. The use according to claim 36 or 37, wherein the mammal is a human.

39. The use according to any one of claims 36 to 38, wherein the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, myeloma, breast cancer, prostate cancer, esophageal cancer, gastric cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, and astrocytoma.

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