T cell receptor targeting Y220C or R175H mutation in P53

Abstract

An isolated or purified T cell receptor (TCR) having antigen specificity for human p53Y220C or human p53R175H is disclosed. Related polypeptides and proteins as well as related nucleic acids, recombinant expression vectors, host cells, populations of host cells and pharmaceutical compositions are also provided. Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are also disclosed.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 492,256, filed March 27, 2023, which is incorporated herein by reference in its entirety.

[0003] Statement regarding federally sponsored research or development

[0004] This invention was carried out with government support by the National Institutes of Health and the National Cancer Institute (NIH), under project number BC010985. The government holds certain rights to this invention.

[0005] By referencing materials incorporated into electronic submissions

[0006] The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this document is incorporated herein by reference in its entirety and is identified as a 116,338-byte XML file named “770310.XML” dated March 22, 2024. Background of the Invention

[0008] Some cancers may have very limited treatment options, especially when they become metastatic and unresectable. Despite advances in treatments such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer, can be poor. Therefore, there is an unmet need for alternative treatments for cancer.

[0009] Brief Overview of the Invention

[0010] One aspect of the present invention provides for human p53 Y220C or person p53 R175H An isolated or purified T-cell receptor (TCR) with an antigen-specific amino acid sequence, wherein the TCR comprises the following amino acid sequences: (1) all SEQ ID NOs: 2-7; (2) all SEQ ID NOs: 16-21; (3) all SEQ ID NOs: 30-35; (4) all SEQ ID NOs: 44-49; (5) all SEQ ID NOs: 58-63; (6) all SEQ ID NOs: 72-77; or (7) all SEQ ID NOs: 86-91.

[0011] Other aspects of the invention provide polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions related to the TCR of the invention.

[0012] Other aspects of the invention provide methods for detecting the presence of cancer in mammals, methods for inducing an immune response against cancer in mammals, and methods for treating or preventing cancer in mammals.

[0013] Another aspect of the invention provides methods for generating host cells expressing TCRs and methods for generating TCRs, polypeptides, or proteins.

[0014] Brief description of several views in the attached diagram

[0015] Figure 1A This is a graph showing the frequency (%) of tetramer-positive cells measured under each culture condition 1, 2, and 3 in Table 1. From HLA-A 02:01 The monomer generates a tetramer and is loaded with a p53 R175H 9-mer peptide analog to capture HLA-A 02:01 Restricted p53 R175H mutant reactive T cells.

[0016] Figure 1B-1E This is a graph showing the percentage of mTCR+CD8+ cells expressing 4-1BB after co-culturing effector cells with target cells. Effector cells are healthy donor PBLs transduced with 4196-C TCR (B), 4196-D TCR (C), 4196-E TCR (D), or TCRs 4196_AV6_ and _BV11-2 (E) (disclosed in US 2022 / 0277352, used as “Comparative TCRs”). Target cells are T2 cells naturally expressing HLA-A2 and have been incubated with WT peptide analogs or mutant p53 R175H 9-mer peptide analogs at specified concentrations.

[0017] Figure 1F It includes Figure 1B-1E The same data shown was redrawn together for comparison.

[0018] Figure 1G This is a graph showing normalized tumor cell counts measured at 4-hour intervals after effector cells and target cells were co-cultured. Effector cells were healthy donor PBLs transduced with a retroviral vector encoding 4196-C TCR, 4196-D TCR, 4196-E TCR, or an unrelated TCR. Target cells were naturally expressing p53 R175H and HLA-A. TYK-nu human ovarian cancer cells at 02:01. P < 0.001.

[0019] Figure 2A The following is a flow cytometry data of CD8 and tetramer obtained after staining TILs from patient 4424, the tetramer being used for... Figure 1A middle.

[0020] Figure 2B This graph shows the percentage of mTCR+CD8+ cells expressing 4-1BB as a marker of T cell activation after co-culturing effector and target cells. Effector cells are healthy donor PBLs transduced with 4424-R175H TCR. Target cells are monkey kidney COS7 cells engineered to express HLA-A2 and incubated with either a WT peptide analog or a mutant p53 R175H 9-mer peptide analog at a specified concentration.

[0021] Figure 3 This is a graph showing normalized tumor cell counts measured at 3-hour intervals after co-culturing effector cells and target cells at a 2:1 effector-to-target ratio. Effector cells were healthy donor PBLs transduced with a retroviral vector encoding 4196-C TCR, 4196-D TCR, 4196-E TCR, 4424-R175H TCR, or 4141-TCR1a2 (another “comparative TCR”) disclosed in US 2022 / 0332785. Target cells were TYK-nu human ovarian cancer cells. Statistical tests were performed using two-way ANOVA. p<0.001.

[0022] Figures 4A-4D This graph shows the percentage of mTCR+CD3+ cells expressing 4-1BB after co-culturing effector and target cells. Effector cells are healthy donor PBLs transduced with 4402-Y220C-B TCR, 4402-Y220C-C TCR, 4402-Y220C-L TCR, or 4343-D TCR (“Comparative TCR”). Target cells are autologous B cells pulsed with WT or mutant peptides at specified concentrations.

[0023] Figure 4E This graph shows the percentage of mTCR+CD3+ cells expressing 4-1BB after co-culturing effector and target cells. Effector cells are healthy donor PBLs transduced with 4402-Y220C-C TCR or 4402-Y220C-L TCR. Target cells are 11th generation DRB1 cells from patient 4402. The absence of 13:03 or the reduced but detectable expression level of DRB1 in the 27th generation. 13:03 Autologous breast tumor organoid cells (see...) Figure 4FTCR-C and L showed that they only recognized 27th generation organoids but not 11th generation organoids, indicating that both TCRs are affected by DRB1. Limitations of 13:03. Target cells pulsed with DMSO were used as a control.

[0024] Figure 4F This shows HLA-DRB1 in the 11th and 27th generations of autologous organoid cells from patient 4402. 15:01 and DRB1 A graph of kilobase-million-transcriptions (TPM) values ​​measured at 13:03.

[0025] Figures 5A-5C The amino acid sequences of nine p53 splice variants are shown in the comparison. SP|P04637|P53_HUMAN(SEQ ID NO: 1); SP|P04637-2|P53_HUMAN(SEQ ID NO: 104); SP|P04637-3|P53_HUMAN(SEQ ID NO: 105); SP|P04637-4|P53_HUMAN(SEQ ID NO: 106);SP|P04637-5|P53_HUMAN(SEQ IDNO: 107);SP|P04637-6|P53_HUMAN(SEQ ID NO: 108);SP|P04637-7|P53_HUMAN(SEQ IDNO: 109);SP|P04637-8|P53_HUMAN(SEQ ID NO: 110) and SP|P04637-9|P53_HUMAN (SEQ IDNO: 111). The alignment of amino acid residues 1 to 120 of SEQ ID NO: 1 is shown in A, the alignment of amino acid residues 121 to 300 of SEQ ID NO: 1 is shown in B, and the alignment of amino acid residues 301 to 393 of SEQ ID NO: 1 is shown in C.

[0026] Figure 6-7 This is shown by transducing healthy donor T cells at a rate of 2 million cells / mouse ( Figure 6 ) or 10 million cells / mouse ( Figure 7 Following administration of a dose of [specific drug name] to adoptive cell transfer (ACT) mice, the average tumor size (mm) measured at specified days was [specific value]. 2(The image is shown.) Healthy donor T cells were independently transduced using: (i) a retroviral vector encoding the 4196-C TCR of Example 4 (“4196 IVS-C”); (ii) an unrelated TCR control (i.e., 4259) (“4259 (unrelated)”); (iii) TCRs 4196_AV12-1_ and _BV6-1 (“12-6”) disclosed in US 2020 / 0277352; (iv) TCRs 4196_AV38-1_ and _BV10-3 (“38-10”) disclosed in US 2020 / 0277352; (v) TCRs 4196_AV6_ and _BV11-2 (“6-11”) disclosed in US 2020 / 0277352; and (vi) TCRs disclosed in US 2020 / 0332785. 4141-TCR1a2 (“4141 1a2”); or (vii) 4141 IVS TCR (“4141IVS”) disclosed in U.S. Patent Application No. 18 / 289,596. Indicates statistical significance.

[0027] Detailed description of the invention

[0028] The tumor protein p53 (also known as "TP53" or "p53") acts as a tumor inhibitor by, for example, regulating cell division. The p53 protein is located in the cell nucleus, where it binds directly to DNA. When DNA is damaged, the p53 protein is involved in determining whether the DNA will be repaired or whether the damaged cell will undergo apoptosis. If the DNA can be repaired, p53 activates other genes to fix the damage. If the DNA cannot be repaired, the p53 protein stops cell division and signals it to undergo apoptosis. By stopping cell division with mutated or damaged DNA, p53 helps prevent tumor development. WT (normal) full-length p53 contains the amino acid sequence of SEQ ID NO: 1.

[0029] Mutations in the p53 protein can reduce or eliminate its tumor-suppressive function. Optionally or additionally, p53 mutations can be gain-of-function mutations that interfere with the function of WT p53 in a dominant-negative manner. Mutated p53 proteins can be expressed in any of a variety of human cancers, such as, for example, cholangiocarcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer (NSCLC), glioblastoma, cervical cancer, head and neck cancer, breast cancer, pancreatic cancer, or bladder cancer.

[0030] One aspect of the present invention provides for human p53 Y220C or person p53 R175HThe amino acid sequence (hereinafter referred to as "mutated p53") is an isolated or purified T-cell receptor (TCR) with antigen specificity. Hereinafter, unless otherwise stated, "TCR" also refers to a functional portion or functional variant of the TCR. Mutations in p53 are defined herein by reference to the amino acid sequence of the full-length WT p53 (SEQ ID NO: 1). Mutations in p53 are described herein by reference to the amino acid residue present at a specific position, followed by the position number, and then the amino acid that is substituted by the specific mutated amino acid residue discussed. A p53 amino acid sequence (e.g., a p53 peptide) may contain fewer amino acid residues than the full-length WT p53 protein. Therefore, while position numbers are defined herein by reference to the full-length WT p53 protein (i.e., SEQ ID NO: 1), it should be understood that the actual positions of the corresponding residues in a particular example of the p53 amino acid sequence may differ. Because the positions are as defined in SEQ ID NO: 1, the term "Y220C" indicates that the tyrosine at position 220 of SEQ ID NO: 1 is replaced by a cysteine; and "R175H" indicates that the arginine at position 175 of SEQ ID NO: 1 is replaced by a histidine. For example, when a specific example of the p53 amino acid sequence is, for example: DRNTFRHSVVVP Y When EPPEVGSDCTTI (SEQ ID NO: 114) (corresponding to the exemplary WT p53 peptide of consecutive amino acid residues 208 to 232 of SEQ ID NO: 1), "Y220C" means that the underlined tyrosine in SEQ ID NO: 114 is replaced by a cysteine, even though the actual position of the underlined tyrosine in SEQ ID NO: 114 is 13. The human p53 amino acid sequence with the Y220C mutation is referred to below as "Y220C" or "p53". Y220C The human p53 amino acid sequence with the R175H mutation will be referred to as "R175H" or "p53" below. R175H As used in this article, "mutated p53" refers to human p53. Y220C or person p53 R175H .

[0031] P53 has nine known splicing variants. The p53 mutations described in this paper are conserved in all nine p53 splicing variants. A comparison of the nine p53 splicing variants shows... Figures 5A-5CTherefore, the TCR of the present invention is antigen-specific for any mutated p53 amino acid sequence encoded by any of the nine p53 splice variants described herein. Because the position is defined by SEQ ID NO: 1, the actual position of the amino acid sequence of a particular splice variant of p53 is defined relative to the corresponding position in SEQ ID NO: 1, and the position defined by SEQ ID NO: 1 may differ from the actual position in the particular splice variant. Thus, for example, a mutation refers to the substitution of an amino acid residue in the amino acid sequence of a particular splice variant of p53 corresponding to a specified position in the 393-amino acid sequence of SEQ ID NO: 1, and it should be understood that the actual position in the splice variant may differ.

[0032] In one aspect of the invention, the TCR is antigen-specific to human p53 with a mutation at position 220 as defined in SEQ ID NO: 1. The p53 mutation at position 220 can be a missense mutation. Therefore, the mutation at position 220 can be a substitution of the native (WT) tyrosine residue present at position 220 with any amino acid residue other than tyrosine. In one aspect of the invention, the TCR is antigen-specific to human p53. Y220C Amino acid sequences are antigen-specific. For example, TCR can target DRNTFRHSVVVP. C EPPEVGSDCTTI (SEQ ID NO: 114) p53 Y220C The amino acid sequence is antigen-specific. In one aspect of the invention, TCR targets DRNTFRHSVVVP. Y The wild-type human p53 amino acid sequence of EPPEVGSDCTTI (SEQ ID NO: 115) is not antigen-specific.

[0033] In one aspect of the invention, the TCR is antigen-specific to human p53 with a mutation at position 175 as defined in SEQ ID NO: 1. The p53 mutation at position 175 can be a missense mutation. Therefore, the mutation at position 175 can be a substitution of the native (WT) arginine residue present at position 175 with any amino acid residue other than arginine. In one aspect of the invention, the TCR is antigen-specific to human p53. R175H Amino acid sequences are antigen-specific. For example, TCR can target HMTEVVR. H People with C (SEQ ID NO: 112) p53 R175H The amino acid sequence is antigen-specific. In one aspect of the invention, TCR targets HMTEVVR. R The wild-type human p53 amino acid sequence of C (SEQ ID NO: 113) does not have antigen specificity.

[0034] In one aspect of the invention, the TCR of the present invention is capable of recognizing mutated p53 in an HLA (human leukocyte antigen) molecule-dependent manner. As used herein, "HLA molecule-dependent manner" means that the TCR, upon binding to mutated p53 in a background of HLA molecules expressed by a patient from whom the TCR was isolated, elicits an immune response. The TCR of the present invention is capable of recognizing mutated p53 presented by applicable HLA molecules and, in addition to mutated p53, can also bind to HLA molecules.

[0035] In one aspect of the invention, the TCR of the present invention is capable of recognizing Y220C presented by HLA class II molecules. In this respect, the TCR can elicit an immune response upon binding to Y220C in the context of HLA class II molecules. The TCR of the present invention is capable of recognizing Y220C presented by HLA class II molecules and, in addition to Y220C, can also bind to other HLA class II molecules.

[0036] In one aspect of the invention, HLA class II molecules are HLA-DR heterodimers. HLA-DR heterodimers are cell surface receptors comprising α and β chains. The HLA-DR α chain is encoded by the HLA-DRRA gene. The HLA-DR β chain is encoded by the HLA-DRB1, HLA-DRB3, HLA-DRB4, or HLA-DRB5 gene. Examples of molecules encoded by the HLA-DRB1 gene include, but are not limited to, HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR16, and HLA-DR17. The HLA-DRB3 gene encodes HLA-DR52. The HLA-DRB4 gene encodes HLA-DR53. The HLA-DRB5 gene encodes HLA-DR51.

[0037] On the one hand, the α chain of HLA class II molecules is composed of HLA-DRA1 01:01 allele expression. On the one hand, the β chain of HLA class II molecules is composed of HLA-DRB3. 02:02 allele expression. In one aspect of the invention, the HLA class II molecule is an HLA-DRB3:HLA-DRA heterodimer. In a preferred aspect, the HLA class II molecule is HLA-DRA1. 01: 01 chain and HLA-DRB3 02: A heterodimer of the 02 chain. In a particularly preferred aspect, the mutated p53 is Y220C and the HLA class II molecule is HLA-DRA1. 01: 01 chain and HLA-DRB3 02: The heterodimer of the 02 chain.

[0038] On the one hand, the α chain of HLA class II molecules is composed of HLA-DRA1 01:01 allele expression. On the one hand, the β chain of HLA class II molecules is composed of HLA-DRB1. 13:03 Allele expression. In one aspect of the invention, the HLA class II molecule is an HLA-DRB1:HLA-DRA heterodimer. In a preferred aspect, the HLA class II molecule is HLA-DRA1. 01: 01 chain and HLA-DRB1 13:03 heterodimer of the chain. In a particularly preferred aspect, the mutated p53 is Y220C and the HLA class II molecule is HLA-DRA1. 01: 01 chain and HLA-DRB1 13:03 heterodimer of the chain.

[0039] In one aspect of the invention, the TCR of the present invention is capable of recognizing R175H presented by HLA class I molecules. In this respect, the TCR can elicit an immune response upon binding to R175H in the context of HLA class I molecules. The TCR of the present invention is capable of recognizing R175H presented by HLA class I molecules and, in addition to R175H, can also bind to HLA class I molecules.

[0040] In embodiments of the present invention, HLA class I molecules are HLA-A molecules. HLA-A molecules are heterodimers of the α chain and β2-microglobulin. The HLA-A α chain can be encoded by the HLA-A gene. The β2-microglobulin non-covalently binds the α1, α2, and α3 domains of the α chain to construct the HLA-A complex. HLA-A molecules can be any HLA-A molecule. In embodiments of the present invention, HLA class I molecules are HLA-A2 molecules. HLA-A2 molecules can be any HLA-A2 molecule. Examples of HLA-A2 molecules may include, but are not limited to, those derived from HLA-A... 02:01, HLA-A 02:02, HLA-A 02:03 Alleles, HLA-A 02:05, HLA-A 02:06, HLA-A 02:07 allele or HLA-A 02:11 alleles encode those. Preferably, HLA class I molecules are encoded by HLA-A. 02:01 Allele encoding.

[0041] The TCR of the present invention can provide any one or more of the numerous advantages, including when expressed by cells used for adoptive cell transfer. Mutated p53 is expressed by cancer cells and not by normal non-cancer cells. Not limited to any particular theory or mechanism, the TCR of the present invention is thought to advantageously target the destruction of cancer cells while minimizing or eliminating the destruction of normal non-cancer cells, thereby reducing (e.g., by minimizing or eliminating) toxicity. Furthermore, the TCR of the present invention can advantageously and successfully treat or prevent mutant p53-positive cancers that are unresponsive to other types of treatments (such as, for example, chemotherapy, surgery, or radiotherapy). Additionally, the TCR of the present invention can provide a high affinity recognition of mutant p53, which can provide the ability to recognize unmanipulated tumor cells (e.g., tumor cells untreated with interferon (IFN)-γ, transfected with a vector encoding mutant p53 and one or both of applicable HLA molecules, pulsed with a p53 peptide having a p53 mutation, or a combination thereof). Mutations in p53 are common in different tumor types. Nearly half of tumors have mutations in p53, about half of which are missense mutations. The R175H mutation is common, affecting approximately 5% of all solid cancer patients. The Y220C mutation is also highly recurrent and affects approximately 0.4% of all cancer patients. Therefore, the TCR of this invention can increase the number of patients who are suitable for immunotherapy.

[0042] As used herein, the phrase “antigen specificity” means that the TCR can specifically bind with high affinity and immunely recognize mutated p53. For example, if approximately 1 x 10 4 Up to 1 x 10 5T cells expressing TCR secrete at least about 200 pg / mL or more (e.g., 200 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL, 10 ng / mL, 100 ng / mL, 500 ng / mL, 1,000 ng / mL, 5,000 ng / mL, 10,000 ng / mL, or the range defined by any two of the foregoing values) after being co-cultured with antigen-negative HLA-positive target cells pulsed with a mutant p53 peptide (e.g., about 0.1 ng / mL to 10,000 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 500 ng / mL, 1,000 ng / mL, or the range defined by any two of the foregoing values). If the TCR contains IFN-γ at concentrations of pg / mL or more, 700 pg / mL or more, 1000 pg / mL or more, 5000 pg / mL or more, 7000 pg / mL or more, 10000 pg / mL or more, 20000 pg / mL or more, or within the range defined by any two of the foregoing values, then the TCR can be considered to have “antigen specificity” for the mutated p53. Cells expressing the TCR of the present invention can also secrete IFN-γ after co-culturing with antigen-negative HLA-positive target cells pulsed with a higher concentration of the mutated p53 peptide.

[0043] Optionally or additionally, TCR can be considered “antigen-specific” to mutated p53 if, compared to the amount of IFN-γ expressed in a negative control, T cells expressing TCR secrete at least twice the amount of IFN-γ after co-culturing with (a) antigen-negative HLA-positive target cells pulsed with a mutant p53 peptide, or with (b) target cells in which a nucleotide sequence encoding a mutant p53 has been introduced such that antigen-negative HLA-positive target cells express the mutant p53. Negative controls may be, for example, (i) antigen-negative HLA-positive target cells pulsed with (a) an unrelated peptide (e.g., some other peptide having a sequence different from the mutated p53), or (b) T cells expressing TCR co-cultured with said target cells that have been introduced with a nucleotide sequence encoding the unrelated peptide, such that the antigen-negative HLA-positive target cells express the unrelated peptide; or (ii) untransduced T cells (e.g., derived from PBMCs that do not express TCR) co-cultured with (a) antigen-negative HLA-positive target cells pulsed with the same concentration of the mutated p53 peptide, or (b) said target cells that have been introduced with a nucleotide sequence encoding the mutated p53, such that the antigen-negative HLA-positive target cells express the mutated p53. IFN-γ secretion can be measured by methods known in the art (e.g., enzyme-linked immunosorbent assay (ELISA)). The concentration of the pulsed peptide may be as described herein with respect to other aspects of the invention.

[0044] Optionally or additionally, if at least twice the number of T cells expressing TCR secrete IFN-γ compared to the number of negative control T cells secreting IFN-γ, then TCR can be considered “antigen-specific” to the mutant p53. This is achieved when the target cells, after co-culturing with (a) antigen-negative HLA-positive target cells pulsed with a mutant p53 peptide, or (b) target cells for which a nucleotide sequence encoding a mutant p53 has been introduced such that antigen-negative HLA-positive target cells express the mutant p53, the TCR can be considered “antigen-specific” to the mutant p53. The concentrations of the peptide and the negative control can be as described herein with respect to other aspects of the invention. The number of cells secreting IFN-γ can be measured by methods known in the art, such as, for example, an enzyme-linked immunospot (ELISPOT) assay.

[0045] Optionally or additionally, if, after co-culturing with target cells that are antigen-negative HLA-positive target cells pulsed with a mutant p53 peptide, or (b) target cells that have had a nucleotide sequence encoding a mutant p53 introduced such that antigen-negative HLA-positive target cells express the mutant p53, the TCR can be considered "antigen-specific" to the mutant p53 if, after co-culturing with such target cells, at least twice as many spots are detected by ELISPOT for T cells expressing the mutant p53, then the TCR can be considered "antigen-specific". The concentrations of the peptide and the negative control can be as described herein with respect to other aspects of the invention.

[0046] Optionally or otherwise, such as as measured by flow cytometry after stimulation with target cells expressing mutant p53, if T cells expressing TCR upregulate one or both of 4-1BB and OX40, then TCR can be considered to have “antigen specificity” for mutant p53.

[0047] One aspect of the present invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as the α chain, β chain, γ chain, δ chain, or combinations thereof of the TCR. The polypeptides of the TCR of the present invention may contain any amino acid sequence, provided that the TCR is antigen-specific to mutated p53.

[0048] In one aspect of the invention, the TCR comprises two polypeptide chains, each containing a variable region, said variable region including complementarity-determining regions (CDRs) 1, 2, and 3 of the TCR. In another aspect of the invention, the TCR comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising α-chain CDR1 (CDR1α), α-chain CDR2 (CDR2α), and α-chain CDR3 (CDR3α), and the second polypeptide chain comprising β-chain CDR1 (CDR1β), β-chain CDR2 (CDR2β), and β-chain CDR3 (CDR3β). In one aspect of the invention, the TCR comprises the following amino acid sequences: (1) all SEQ ID NOs: 2-7 (4196-C TCR); (2) all SEQ ID NOs: 16-21 (4196-D TCR); (3) all SEQ ID NOs: 30-35 (4196-E TCR); (4) all SEQ ID NOs: 44-49 (4124-R175H TCR); (5) all SEQ ID NOs: 58-63 (4402-Y220C-B TCR); (6) all SEQ ID NOs: 72-77 (4402-Y220C-C TCR); or (7) all SEQ ID NOs: 86-91 (4402-Y220C-L TCR). Each of the aforementioned seven amino acid sequences in this paragraph lists the six CDR regions of each of the seven different TCRs that are antigen-specific to mutated human p53. The six amino acid sequences in each set correspond to CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the TCR, respectively.

[0049] The TCR may contain any one or more of the following amino acid sequences: SEQ ID NO: 2-7, 16-21, 30-35, 44-49, 58-63, 72-77, and 86-91. In one aspect of the invention, the TCR contains a response to human p53. Y220C or person p53 R175H An isolated or purified T-cell receptor (TCR) with an antigen-specific amino acid sequence, wherein the TCR comprises the following amino acid sequences: (1) all SEQ ID NOs: 2-7; (2) all SEQ ID NOs: 16-21; (3) all SEQ ID NOs: 30-35; (4) all SEQ ID NOs: 44-49; (5) all SEQ ID NOs: 58-63; (6) all SEQ ID NOs: 72-77; or (7) all SEQ ID NOs: 86-91.

[0050] In one aspect of the invention, the TCR comprises an α-chain variable region amino acid sequence and a β-chain variable region amino acid sequence, which together comprise one of the sets of CDRs listed above. In this regard, the TCR may contain the following amino acid sequences: (1) SEQ ID NO: 8 and 9 (4196-C TCR); (2) SEQ ID NO: 10 and 11 (4196-C TCR); (3) SEQ ID NO: 22 and 23 (4196-D TCR); (4) SEQ ID NO: 24 and 25 (4196-D TCR); (5) SEQ ID NO: 36 and 37 (4196-ETCR); (6) SEQ ID NO: 38 and 39 (4196-E TCR); (7) SEQ ID NO: 50 and 51 (4424-R175H TCR); (8) SEQ ID NO: 52 and 53 (4424-R175H TCR); (9) SEQ ID NO: 64 and 65 (4402-Y220C-B TCR); (10) SEQ ID NO: 66 and 67 (4402-Y220C-B TCR); (11) SEQ ID NO: 78 and 79 (4402-Y220C-CTCR); (12) SEQ ID NO: 80 and 81 (4402-Y220C-C TCR); (13) SEQ ID NO: 92 and 93 (4402-Y220C-L TCR); or (14) SEQ ID NO: 94 and 95 (4402-Y220C-L TCR). Each of the aforementioned amino acid sequence sets in this paragraph lists two variable regions of each of the seven different TCRs that are antigen-specific to mutated human p53. The two amino acid sequences in each set correspond to the variable regions of the α chain and β chain of the TCR, respectively.

[0051] The TCR may, for example, contain any one or more amino acid sequences of SEQ ID NO: 8, 9, 10, 11, 22, 23, 24, 25, 36, 37, 38, 39, 50, 51, 52, 53, 64, 65, 66, 67, 78, 79, 80, 81, 92, 93, 94, and 95. In one aspect of the invention, the TCR comprises the following amino acid sequences: (1) SEQ ID NO: 8; (2) SEQ ID NO: 9; (3) SEQ ID NO: 8 and 9; (4) SEQ ID NO: 10; (5) SEQ ID NO: 11; (6) SEQ ID NO: 10 and 11; (7) SEQ ID NO: 22; (8) SEQ ID NO: 23; (9) SEQ ID NO: 22 and 23; (10) SEQ ID NO: 24; (11) SEQ ID NO: 25; (12) SEQ ID NO: 24 and 25; (13) SEQ ID NO: 36; (14) SEQ ID NO: 37; (15) SEQ ID NO: 36 and 37; (16) SEQ ID NO: 38; (17) SEQ ID NO: 39; (18) SEQ ID NO: 38 and 39; (19) SEQ ID NO: 39; NO: 50; (20) SEQ ID NO: 51; (21) SEQ ID NO: 50 and 51; (22) SEQ ID NO: 52; (23) SEQ ID NO: 53; (24) SEQ ID NO: 52 and 53; (25) SEQ ID NO: 64; (26) SEQ ID NO: 65; (27) SEQ ID NO: 64 and 65; (28) SEQ ID NO: 66; (29) SEQ ID NO: 67; (30) SEQ ID NO: 66 and 67; (31) SEQ ID NO: 78; (32) SEQ ID NO: 79; (33) SEQ ID NO: 78 and 79; (34) SEQ ID NO: 80; (35) SEQ ID NO: 81; (36) SEQ ID NO: 80 and 81; (37) SEQ ID NO: 92; (38) SEQ ID NO: 93; (39) SEQ ID NO: 92 and 93; (40) SEQ ID NO: 94; (41) SEQ ID NO: 95; or (42) SEQ ID NO: 94 and 95.

[0052] The TCR of the present invention may also include a constant region. The constant region may be derived from any suitable species, such as, for example, human or mouse. In one aspect of the invention, the TCR also includes a mouse constant region. As used herein, the terms “mouse” or “human” when referring to the TCR or any component of a TCR described herein (e.g., complementarity-determining region (CDR), variable region, constant region, α chain, and / or β chain) mean a TCR (or a component thereof) derived from mouse or human, respectively, i.e., derived from or formerly expressed by mouse T cells or human T cells, respectively. In one aspect of the invention, the TCR may include a mouse α chain constant region and a mouse β chain constant region. The mouse α chain constant region may be modified or unmodified. Modified mouse α chain constant regions may be, for example, cysteine-substituted, LVL-modified, or cysteine-substituted and LVL-modified, as described in, for example, U.S. Patent Application No. 10,174,098. The mouse β chain constant region may be modified or unmodified.

[0053] The modified mouse β-chain constant region can be, for example, cysteine-substituted, as described in U.S. Patent No. 10,174,098. In one aspect of the invention, the TCR comprises a cysteine-substituted, LVL-modified mouse α-chain constant region containing the amino acid sequence of SEQ ID NO: 100 or 101. In another aspect of the invention, the TCR comprises a cysteine-substituted mouse β-chain constant region containing the amino acid sequence of SEQ ID NO: 102.

[0054] In one aspect of the invention, the TCR of the present invention may comprise an α chain and a β chain. The α chain of the TCR may comprise a variable region and a constant region of the α chain. This type of α chain can pair with any β chain of the TCR. The β chain may comprise a variable region and a constant region of the β chain.

[0055] In one aspect of the present invention, the TCR may comprise the following amino acid sequences: (1) SEQ ID NO: 12 and 13 (4196-C TCR); (2) SEQ ID NO: 14 and 15 (4196-C TCR); (3) SEQ ID NO: 26 and 27 (4196-D TCR); (4) SEQ ID NO: 28 and 29 (4196-D TCR); (5) SEQ ID NO: 40 and 41 (4196-E TCR); (6) SEQ ID NO: 42 and 43 (4196-E TCR); (7) SEQ ID NO: 54 and 55 (4424-R175H TCR); (8) SEQ ID NO: 56 and 57 (4424-R175H); (9) SEQ ID NO: 68 and 69 (4402-Y220C-B TCR); (10) SEQ ID NO: 12 and 13 (4196-C TCR); (11) SEQ ID NO: 12 and 13 (4196-D TCR); (2) SEQ ID NO: 14 and 15 (4196-C TCR); (3) SEQ ID NO: 26 and 27 (4196-D TCR); (4) SEQ ID NO: 28 and 29 (4196-D TCR); (5) SEQ ID NO: 40 and 41 (4196-E TCR); (6) SEQ ID NO: 42 and 43 (4196-E TCR); (7) SEQ ID NO: 54 and 55 (4424-R175H TCR); (8) SEQ ID NO: 56 and 57 (4424-R175H); (9) SEQ ID NO: 68 and 69 (4402-Y (11) SEQ ID NO: 70 and 71 (4402-Y220C-B TCR); (12) SEQ ID NO: 82 and 83 (4402-Y220C-C TCR); (13) SEQ ID NO: 96 and 97 (4402-Y220C-L TCR); or (14) SEQ ID NO: 98 and 99 (4402-Y220C-L TCR). Each of the aforementioned amino acid sequence sets in this paragraph lists the α and β chains of each of the seven different TCRs that are antigen-specific to mutated human p53. The two amino acid sequences in each set correspond to the α and β chains of the TCR, respectively.

[0056] The TCR may contain any one or more amino acid sequences of SEQ ID NO: 12, 13, 14, 15, 26, 27, 28, 29, 40, 41, 42, 43, 54, 55, 56, 57, 68, 69, 70, 71, 82, 83, 84, 85, 96, 97, 98, and 99. In one aspect of the invention, the TCR comprises the following amino acid sequences: (1) SEQ ID NO: 12; (2) SEQ ID NO: 13; (3) SEQ ID NO: 12 and 13; (4) SEQ ID NO: 14; (5) SEQ ID NO: 15; (6) SEQ ID NO: 14 and 15; (7) SEQ ID NO: 26; (8) SEQ ID NO: 27; (9) SEQ ID NO: 26 and 27; (10) SEQ ID NO: 28; (11) SEQ ID NO: 29; (12) SEQ ID NO: 28 and 29; (13) SEQ ID NO: 40; (14) SEQ ID NO: 41; (15) SEQ ID NO: 40 and 41; (16) SEQ ID NO: 42; (17) SEQ ID NO: 43; (18) SEQ ID NO: 42 and 43; (19) SEQ ID NO: 54; (20) SEQ ID NO: 55; (21) SEQ ID NO: 54 and 55; (22) SEQ ID NO: 56; (23) SEQ ID NO: 57; (24) SEQ ID NO: 56 and 57; (25) SEQ ID NO: 68; (26) SEQ ID NO: 69; (27) SEQ ID NO: 68 and 69; (28) SEQ ID NO: 70; (29) SEQ ID NO: 71; (30) SEQ ID NO: 70 and 71; (31) SEQ ID NO: 82; (32) SEQ ID NO: 83; (33) SEQ ID NO: 82 and 83; (34) SEQ ID NO: 84; (35) SEQ ID NO: 85; (36) SEQ ID NO: 84 and 85; (37) SEQ ID NO: 96; (38) SEQ ID NO: 97; (39) SEQ ID NO: 96 and 97; (40) SEQ ID NO: 98; (41) SEQ ID NO: 99; or (42) SEQ ID NO: 98 and 99.

[0057] Included within the scope of this invention are functional variants of the TCRs of the present invention described herein. As used herein, the term "functional variant" refers to a TCR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parental TCR, polypeptide, or protein, and that retains the biological activity of the TCR, polypeptide, or protein from which it is a variant. Functional variants include, for example, those variants of the TCR, polypeptide, or protein (parental TCR, polypeptide, or protein) described herein that retain the ability to specifically bind mutated p53 to a degree similar to, the same as, or greater than that of the parental TCR, polypeptide, or protein, to which the parental TCR has antigen specificity, or to which the parental polypeptide or protein specifically binds mutated p53. With respect to the parental TCR, polypeptide, or protein, the functional variant may, for example, be identical to the parental TCR, polypeptide, or protein in at least about 30%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more amino acid sequences, respectively.

[0058] Functional variants may, for example, comprise the amino acid sequence of a parental TCR, polypeptide, or protein having at least one conserved amino acid substitution. Conserved amino acid substitutions are known in the art and include the substitution of one amino acid having certain physical and / or chemical properties with another amino acid having the same chemical or physical properties. For example, a conserved amino acid substitution may be the substitution of one acidic amino acid for another (e.g., Asp or Glu), the substitution of one amino acid with a nonpolar side chain for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), the substitution of one basic amino acid for another basic amino acid (Lys, Arg, etc.), the substitution of one amino acid with a polar side chain for another amino acid with a polar side chain, etc. (Asn, Cys, Gln, Ser, Thr, Tyr, etc.).

[0059] Optionally or additionally, the functional variant may comprise the amino acid sequence of the parental TCR, peptide, or protein having at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parental TCR, peptide, or protein.

[0060] TCRs, peptides, or proteins may consist essentially of one or more specific amino acid sequences as described herein, such that other components of the TCR, peptide, or protein (e.g., other amino acids) do not substantially alter the biological activity of the TCR, peptide, or protein.

[0061] This invention also provides polypeptides comprising any functional portion of a TCR described herein. As used herein, the term "polypeptide" includes oligopeptides, which are single-chain amino acids linked by one or more peptide bonds.

[0062] Regarding the polypeptides of the present invention, the functional portion can be any continuous amino acid fraction comprising a portion of the TCR, as long as the functional portion specifically binds to mutated p53. The term "functional portion," when used in reference to the TCR, refers to any portion or fragment of the TCR of the present invention that retains its biological activity as part of the TCR (parental TCR). The functional portion includes, for example, those portions of the TCR that retain the ability to specifically bind to mutated p53 (e.g., in an applicable HLA-dependent manner), or the ability to detect, treat, or prevent cancer, to a degree similar to, the same as, or greater than that of the parental TCR. Regarding the parental TCR, the functional portion may comprise, for example, about 10%, about 25%, about 30%, about 50%, about 70%, about 80%, about 90%, about 95%, or more of the parental TCR.

[0063] The functional moiety may contain additional amino acids at the amino or carboxyl terminus, or at both ends, that are not present in the amino acid sequence of the parental TCR. Ideally, the additional amino acids do not interfere with the biological function of the functional moiety, such as specifically binding to mutated p53; and / or having the ability to detect, treat, or prevent cancer. More ideally, the additional amino acids enhance the biological activity compared to the parental TCR.

[0064] The polypeptide may contain functional portions of one or both of the α and β chains of the TCR of the present invention, for example, functional portions of one or more of the CDR1, CDR2, and CDR3 of the variable regions of the α and / or β chains of the TCR of the present invention. In one aspect of the present invention, the polypeptide comprises the following amino acid sequences: (1) all SEQ ID NOs: 2-7; (2) all SEQ ID NOs: 16-21; (3) all SEQ ID NOs: 30-35; (4) all SEQ ID NOs: 44-49; (5) all SEQ ID NOs: 58-63; (6) all SEQ ID NOs: 72-77; or (7) all SEQ ID NOs: 86-91. The polypeptide may contain any one or more of the following amino acid sequences: SEQ ID NOs: 2-7, 16-21, 30-35, 44-49, 58-63, 72-77, and 86-91.

[0065] In one aspect of the invention, the polypeptide of the invention may comprise, for example, a variable region of the TCR of the invention containing a combination of the CDR regions listed above. In this respect, the polypeptide may comprise, for example, the following amino acid sequences: (1) SEQ ID NO: 8 and 9; (2) SEQ ID NO: 10 and 11; (3) SEQ ID NO: 22 and 23; (4) SEQ ID NO: 24 and 25; (5) SEQ ID NO: 36 and 37; (6) SEQ ID NO: 38 and 39; (7) SEQ ID NO: 50 and 51; (8) SEQ ID NO: 52 and 53; (9) SEQ ID NO: 64 and 65; (10) SEQ ID NO: 66 and 67; (11) SEQ ID NO: 78 and 79; (12) SEQ ID NO: 80 and 81; (13) SEQ ID NO: 92 and 93; or (14) SEQ ID NO: 94 and 95. The polypeptide may, for example, contain one or more of the amino acid sequences of SEQ ID NO:8, 9, 10, 11, 22, 23, 24, 25, 36, 37, 38, 39, 50, 51, 52, 53, 64, 65, 66, 67, 78, 79, 80, 81, 92, 93, 94, and 95.In one aspect of the invention, the polypeptide comprises the following amino acid sequences: (1) SEQ ID NO: 8; (2) SEQ ID NO: 9; (3) SEQ ID NO: 8 and 9; (4) SEQ ID NO: 10; (5) SEQ ID NO: 11; (6) SEQ ID NO: 10 and 11; (7) SEQ ID NO: 22; (8) SEQ ID NO: 23; (9) SEQ ID NO: 22 and 23; (10) SEQ ID NO: 24; (11) SEQ ID NO: 25; (12) SEQ ID NO: 24 and 25; (13) SEQ ID NO: 36; (14) SEQ ID NO: 37; (15) SEQ ID NO: 36 and 37; (16) SEQ ID NO: 38; (17) SEQ ID NO: 39; (18) SEQ ID NO: 38 and 39; (19) SEQ ID NO: 50; (20) SEQ ID NO: 51; (21) SEQ ID NO: 50 and 51; (22) SEQ ID NO: 52; (23) SEQ ID NO: 53; (24) SEQ ID NO: 52 and 53; (25) SEQ ID NO: 64; (26) SEQ ID NO: 65; (27) SEQ ID NO: 64 and 65; (28) SEQ ID NO: 66; (29) SEQ ID NO: 67; (30) SEQ ID NO: 66 and 67; (31) SEQ ID NO: 78; (32) SEQ ID NO: 79; (33) SEQ ID NO: 78 and 79; (34) SEQ ID NO: 80; (35) SEQ ID NO: 81; (36) SEQ ID NO: 80 and 81; (37) SEQ ID NO: 92; (38) SEQ ID NO: 93; (39) SEQ ID NO: 92 and 93; (40) SEQ ID NO: 94; (41) SEQ ID NO: 95; or (42) SEQ ID NO: 94 and 95.

[0066] In one aspect of the invention, the polypeptide of the invention may further comprise the constant region of the TCR of the invention listed above. In this respect, the polypeptide may comprise, for example, an amino acid sequence of (i) one of SEQ ID NO:100-102 or (ii) one of SEQ ID NO:102 and one of SEQ ID NO:100 and 101.

[0067] In one aspect of the invention, the polypeptide of the invention may comprise the α chain and β chain of the TCR of the invention. In this respect, the polypeptide may comprise, for example, the following amino acid sequences: (1) SEQ ID NO: 12 and 13; (2) SEQ ID NO: 14 and 15; (3) SEQ ID NO: 26 and 27; (4) SEQ ID NO: 28 and 29; (5) SEQ ID NO: 40 and 41; (6) SEQ ID NO: 42 and 43; (7) SEQ ID NO: 54 and 55; (8) SEQ ID NO: 56 and 57; (9) SEQ ID NO: 68 and 69; (10) SEQ ID NO: 70 and 71; (11) SEQ ID NO: 82 and 83; (12) SEQ ID NO: 84 and 85; (13) SEQ ID NO: 96 and 97; or (14) SEQ ID NO: 98 and 99. The polypeptide may contain any one or more of the amino acid sequences of SEQ ID NO: 12, 13, 14, 15, 26, 27, 28, 29, 40, 41, 42, 43, 54, 55, 56, 57, 68, 69, 70, 71, 82, 83, 84, 85, 96, 97, 98 and 99.In one aspect of the invention, the polypeptide comprises the following amino acid sequences: (1) SEQ ID NO: 12; (2) SEQ ID NO: 13; (3) SEQ ID NO: 12 and 13; (4) SEQ ID NO: 14; (5) SEQ ID NO: 15; (6) SEQ ID NO: 14 and 15; (7) SEQ ID NO: 26; (8) SEQ ID NO: 27; (9) SEQ ID NO: 26 and 27; (10) SEQ ID NO: 28; (11) SEQ ID NO: 29; (12) SEQ ID NO: 28 and 29; (13) SEQ ID NO: 40; (14) SEQ ID NO: 41; (15) SEQ ID NO: 40 and 41; (16) SEQ ID NO: 42; (17) SEQ ID NO: 43; (18) SEQ ID NO: 42 and 43; (19) SEQ ID NO: 54; (20) SEQ ID NO: 55; (21) SEQ ID NO: 54 and 55; (22) SEQ ID NO: 56; (23) SEQ ID NO: 57; (24) SEQ ID NO: 56 and 57; (25) SEQ ID NO: 68; (26) SEQ ID NO: 69; (27) SEQ ID NO: 68 and 69; (28) SEQ ID NO: 70; (29) SEQ ID NO: 71; (30) SEQ ID NO: 70 and 71; (31) SEQ ID NO: 82; (32) SEQ ID NO: 83; (33) SEQ ID NO: 82 and 83; (34) SEQ ID NO: 84; (35) SEQ ID NO: 85; (36) SEQ ID NO: 84 and 85; (37) SEQ ID NO: 96; (38) SEQ ID NO: 97; (39) SEQ ID NO: 96 and 97; (40) SEQ ID NO: 98; (41) SEQ ID NO: 99; or (42) SEQ ID NO: 98 and 99.

[0068] Another aspect of the invention provides proteins comprising any functional portion of the TCRs of the invention as described herein. "Protein" means a molecule comprising one or more polypeptide chains. On one hand, the protein of the present invention may comprise: a first polypeptide chain and a second polypeptide chain, wherein: (1) the first polypeptide chain comprises all amino acid sequences of SEQ ID NO: 2-4; (2) the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 5-7; (3) the first polypeptide chain comprises all amino acid sequences of SEQ ID NO: 2-4 and the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 5-7; (4) the first polypeptide chain comprises all amino acid sequences of SEQ ID NO: 16-18; (5) the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 19-21; (6) the first polypeptide chain comprises all amino acid sequences of SEQ ID NO: 16-18 and the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 19-21; (7) the first polypeptide chain comprises all amino acid sequences of SEQ ID NO: 30-32; (8) the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 33-35; (9) the first polypeptide chain comprises all amino acid sequences of SEQ ID NO: 30-32; (1) the first polypeptide chain comprises all amino acid sequences of SEQ ID NO: 33-35; (10) the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 33-35; (11) the first polypeptide chain comprises all amino acid sequences of SEQ ID NO: 33-35; (12) the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 33-35; (13) the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 33-35; (14) the first polypeptide chain comprises all amino acid sequences of SEQ ID NO: 33-35; (15) the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 33-35; (16) the second polypeptide chain comprises all amino acid sequences of SEQ ID NO: 33-35; (16) the second polypeptide chain comprises all amino acid sequences of SEQ ID NO (10) The first polypeptide chain contains all amino acid sequences of SEQ ID NO: 44-46; (11) The second polypeptide chain contains all amino acid sequences of SEQ ID NO: 47-49; (12) The first polypeptide chain contains all amino acid sequences of SEQ ID NO: 44-46 and the second polypeptide chain contains all amino acid sequences of SEQ ID NO: 47-49; (13) The first polypeptide chain contains all amino acid sequences of SEQ ID NO: 58-60; (14) The second polypeptide chain contains all amino acid sequences of SEQ ID NO: 61-63; (15) The first polypeptide chain contains all amino acid sequences of SEQ ID NO: 58-60 and the second polypeptide chain contains all amino acid sequences of SEQ ID NO: 61-63; (16) The first polypeptide chain contains all amino acid sequences of SEQ ID NO: 72-74; (17) The second polypeptide chain contains all amino acid sequences of SEQ ID NO: 72-74; (18) The first polypeptide chain contains all the amino acid sequences of SEQ ID NO: 72-74 and the second polypeptide chain contains all the amino acid sequences of SEQ ID NO: 75-77; (19) The first polypeptide chain contains all the amino acid sequences of SEQ ID NO: 86-88; (20) The second polypeptide chain contains all the amino acid sequences of SEQ ID NO: 89-91;Or (21) the first polypeptide chain contains the amino acid sequence of all SEQ ID NO: 86-88 and the second polypeptide chain contains the amino acid sequence of all SEQ ID NO: 89-91.

[0069] In one aspect of the invention, the protein comprises a first polypeptide chain and a second polypeptide chain, wherein: (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; (2) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10; (5) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 11; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 11; (7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22; (8) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 23; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 23; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 24; (11) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 24. (12) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 24 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 25; (13) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 36; (14) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 37; (15) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 36 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 37; (16) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 38; (17) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 39; (18) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 38 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 39; (19) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 50; (20) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 51; (21) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 25. (22) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 52; (23) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 53; (24) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 52 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 53;(25) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 64; (26) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 65; (27) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 64 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 65; (28) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 66; (29) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 67; (30) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 66 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 67; (31) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 78; (32) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 79; (33) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 78 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 79; (34) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 80; (35) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: (36) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 80 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 81; (37) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 92; (38) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 93; (39) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 92 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 93; (40) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 94; (41) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 95; or (42) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 94 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 95.

[0070] In one aspect of the invention, the protein comprises a first polypeptide chain and a second polypeptide chain, wherein: (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 12; (2) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 12 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14; (5) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 15; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 15; (7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 26; (8) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 26 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 12. (11) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 29; (12) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 28 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 29; (13) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 40; (14) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 41; (15) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 40 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 41; (16) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 42; (17) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 43; (18) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 42 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 43; (19) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 54; (20) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 54. (21) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 54 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 55; (22) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 56; (23) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 57; (24) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 56 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 57;(25) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 68; (26) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 69; (27) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 68 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 69; (28) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 70; (29) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 71; (30) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 70 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 71; (31) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 82; (32) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 83; (33) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 82 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 83; (34) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 84; (35) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: (36) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 84 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 85; (37) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 96; (38) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 97; (39) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 96 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 97; (40) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 98; (41) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 99; or (42) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 98 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 99.

[0071] The protein of the present invention may be a TCR. Optionally, if the first polypeptide chain and / or the second polypeptide chain of the protein further comprises other amino acid sequences, such as amino acid sequences encoding immunoglobulins or portions thereof, the protein of the present invention may be a fusion protein. In this regard, one aspect of the present invention also provides a fusion protein comprising at least one polypeptide of the present invention described herein and at least one other polypeptide. The other polypeptide may exist as a separate polypeptide of the fusion protein, or may exist as a polypeptide expressed in-frame (tandemly) with one of the polypeptides of the present invention described herein. The other polypeptide may encode any peptide molecule or protein molecule or portions thereof, including but not limited to immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules (e.g., CD1a, CD1b, CD1c, CD1d), etc.

[0072] The fusion protein may comprise one or more copies of the polypeptide of the present invention and / or one or more copies of other polypeptides. For example, the fusion protein may comprise one, two, three, four, five or more copies of the polypeptide of the present invention and / or other polypeptides. Suitable methods for preparing the fusion protein are known in the art, including, for example, recombinant methods.

[0073] In some aspects of the invention, the TCRs, polypeptides, and proteins of the present invention can be expressed as single proteins comprising a linker peptide connecting the α and β chains. In this respect, the TCRs, polypeptides, and proteins of the present invention may also comprise a linker peptide. The linker peptide can advantageously facilitate the expression of recombinant TCRs, polypeptides, and / or proteins in host cells. The linker peptide can comprise any suitable amino acid sequence. For example, the linker peptide can comprise the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 103). When a construct comprising the linker peptide is expressed by a host cell, the linker peptide can be cleaved to produce separate α and β chains. In one aspect of the invention, the TCR, polypeptide, or protein can comprise an amino acid sequence containing a full-length α chain, a full-length β chain, and a linker peptide located between the α and β chains.

[0074] In some aspects, the TCRs, peptides, or proteins disclosed herein comprise α-chains and / or β-chains as disclosed herein, which contain a signal peptide. In some aspects, the sequence of any α-chain and / or β-chain signal peptide disclosed herein comprises a leucine, lysine, alanine, or histidine residue replacing the second wild-type residue.

[0075] In some respects, the TCRs, peptides, or proteins disclosed herein contain mature versions of the α-chain and / or β-chain disclosed herein that lack a signal peptide.

[0076] The protein of the present invention may be a recombinant antibody or its antigen-binding moiety, comprising at least one polypeptide of the present invention as described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one polypeptide chain of the polypeptide of the present invention and an antibody or its antigen-binding moiety. The polypeptide of the antibody or its antigen-binding moiety may be a heavy chain, a light chain, a variable or constant region of a heavy or light chain, a single-chain variable fragment (scFv), or an Fc, Fab, or F(ab)2' fragment of an antibody, etc. The polypeptide chain of the antibody or its antigen-binding moiety may exist as a separate polypeptide of a recombinant antibody. Optionally, the polypeptide chain of the antibody or its antigen-binding moiety may exist as a polypeptide expressed in (tandem) with the polypeptide frame of the present invention. The polypeptide of the antibody or its antigen-binding moiety may be a polypeptide of any antibody or any antibody fragment (including any antibody and antibody fragment described herein).

[0077] The TCRs, peptides, and proteins of the present invention can have any length and contain any number of amino acids, as long as the TCR, peptide, or protein retains its biological activity, such as the ability to specifically bind to mutated p53; detect cancer in mammals; or treat or prevent cancer in mammals. For example, the length of the peptide can range from about 50 to about 5000 amino acids, for example, lengths of 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acids. In this respect, the peptides of the present invention also include oligopeptides.

[0078] The TCRs, polypeptides, and proteins of the present invention may contain synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, leucine, α-aminodecanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydroindole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornene)carboxylic acid, α, γ-Diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0079] The TCRs, polypeptides, and proteins of the present invention can be, for example, glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, via, for example, disulfide cyclization or conversion to an acid addition salt and / or optionally dimerized, polymerized, or conjugated.

[0080] The TCRs, peptides, and proteins (including any functional portions or functional variants thereof) of the present invention described herein can also be considered as soluble TCR components of bispecific engager TCR fusion proteins (e.g., IMMTAC, an immune mobilization monoclonal TCR molecule against cancer). Bispecific engager TCR fusion proteins have two components. One component comprises a soluble TCR. The other component comprises an anti-CD3 effector. The anti-CD3 effector can be any molecule that binds to CD3 molecules on T cells and activates a T cell immune response. For example, the anti-CD3 effector can be an anti-CD3 antibody or an anti-CD3 antibody fragment. Bispecific engager TCR fusion proteins A soluble TCR component binds to target antigens presented on the surface of cancer cells by HLA molecules. An anti-CD3 effector component binds to CD3 molecules on the surface of T cells. The binding of these components of the bispecifically linked TCR fusion protein triggers T cell activation and recruitment and redirects T cell killing to tumor cells. One aspect of the invention provides a bispecifically linked TCR fusion protein comprising: (i) any TCR, polypeptide, and protein of the invention described herein (including any functional portions or functional variants thereof) and (ii) an anti-CD3 effector. Hereinafter, unless otherwise stated, references to “protein” also include the bispecifically linked TCR fusion protein described herein.

[0081] The TCRs, peptides, and / or proteins of the present invention can be obtained by methods known in the art, such as de novo synthesis. Alternatively, the peptides and proteins can be recombinantly generated using standard recombinant methods for nucleic acids as described herein. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Optionally, the TCRs, peptides, and / or proteins described herein can be synthesized from any of a variety of commercial entities. In this respect, the TCRs, peptides, and proteins of the present invention can be synthetic, recombinant, isolated, and / or purified.

[0082] One aspect of the invention provides nucleic acids comprising a nucleotide sequence encoding any TCR, polypeptide, or protein described herein. As used herein, “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide bonds, such as phosphophosphate bonds or thiophosphate bonds, rather than phosphodiester bonds between the nucleotides of an unmodified oligonucleotide. In one aspect, nucleic acids comprise complementary DNA (cDNA). Generally, it is preferred that nucleic acids do not contain any insertions, deletions, inversions, and / or substitutions. However, as discussed herein, in certain circumstances it may be suitable for nucleic acids to contain one or more insertions, deletions, inversions, and / or substitutions.

[0083] One aspect of the present invention provides isolated or purified nucleic acids comprising, from 5' to 3', a first nucleic acid sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence encode the following amino acid sequences, respectively: SEQ ID NO: 8 and 9; 9 and 8; 10 and 11; 11 and 10; 12 and 13; 13 and 12; 14 and 15; 15 and 14; 22 and 23; 23 and 22; 24 and 25; 25 and 24; 26 and 27; 27 and 26; 28 and 29; 29 and 28; 36 and 37; 37 and 36; 38 and 39; 39 and 38; 40 and 41; 41 and 40; 42 and 43; 43 and 42; 50 and 51; 51 and 50; 52 and 53; 53 and 52; 54 and 55; 55 and 54; 56 and 57; 57 and 56; 64 and 65; 65 and 64; 66 and 67; 67 and 66; 68 and 69; 69 and 68; 70 and 71; 71 and 70; 78 and 79; 79 and 78; 8 or 99 and 98. 0 and 81; 81 and 80; 82 and 83; 83 and 82; 84 and 85; 85 and 84; 92 and 93; 93 and 92; 94 and 95; 95 and 94;

[0084] In one aspect of the invention, the nucleic acid further comprises a third nucleotide sequence inserted between the first and second nucleotide sequences, wherein the third nucleotide sequence encodes a cleavable linker peptide. For example, the cleavable linker peptide may comprise the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 103).

[0085] Preferably, the nucleic acids of the present invention are recombinant. As used herein, the term "recombinant" means (i) a molecule constructed outside a living cell by linking a natural or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule produced by replication of those described in (i) above. For the purposes of this document, replication can be in vitro or in vivo.

[0086] Nucleic acids can be constructed using procedures known in the art based on chemical synthesis and / or enzymatic ligation reactions. See, for example, Green and Sambrook et al., ibid. Nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., phosphate thioester derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the double strands formed during hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N... 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-mercaptocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetic acid, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Optionally, one or more nucleic acids of the present invention can be synthesized by any of a variety of commercial entities.

[0087] In one aspect of the invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. Without being limited to any particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing another codon encoding the same amino acid with a natural codon, but which can be more easily translated by tRNA within the cell, thereby increasing translation efficiency. nucleotide sequence optimization may also reduce secondary mRNA structures that interfere with translation, thereby improving translation efficiency.

[0088] Another aspect of the invention provides nucleic acids comprising nucleotide sequences complementary to the nucleotide sequences of any nucleic acids described herein.

[0089] The nucleic acids of the present invention can be incorporated into recombinant expression vectors. In this regard, one aspect of the present invention provides recombinant expression vectors comprising any of the nucleic acids of the present invention. In one aspect of the present invention, the recombinant expression vector comprises nucleotide sequences encoding an α-chain, a β-chain, and a linker peptide.

[0090] For the purposes of this document, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, allows the host cell to express the mRNA, protein, polypeptide, or peptide. The vectors of this invention are not naturally occurring as a whole. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring, non-naturally occurring internucleotide bonds, or both types of bonds. Preferably, non-naturally occurring or modified nucleotides or internucleotide bonds do not impede transcription or replication of the vector.

[0091] The recombinant expression vector of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for proliferation and amplification, or for expression, or both, such as plasmids and viruses. Vectors can be selected from transposon / transposase series, pUC series (Fermentas LifeSciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, PaloAlto, CA). Phage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, can also be used. Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a transposon or viral vector (e.g., a lentiviral vector or a retroviral vector).

[0092] The recombinant expression vectors of the present invention can be prepared using, for example, standard recombinant DNA techniques described in Green and Sambrook et al., ibid. Circular or linear constructs of the expression vectors can be prepared for inclusion in a functional replication system within a prokaryotic or eukaryotic host cell. The replication system can be derived, for example, from ColE1, 2μ plasmids, λ, SV40, bovine papillomavirus, etc.

[0093] Ideally, recombinant expression vectors contain regulatory sequences, such as transcription and translation start and stop codons, that are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) to which the vector is to be introduced, depending on whether the vector is DNA-based or RNA-based.

[0094] Recombinant expression vectors may include one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include resistance to biocides, such as resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic host cells to provide protrophic forms, etc. Suitable marker genes for use in the expression vectors of this invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0095] Recombinant expression vectors may contain a natural or non-natural promoter operatively linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or a nucleotide sequence complementary to that encoding a TCR, polypeptide, or protein. The selection of the promoter (e.g., strong, weak, inducible, tissue-specific, and developmentally specific) is within the general skill of a person skilled in the art. Similarly, the combination of nucleotide sequences and promoters is also within the skill of a person skilled in the art. Promoters may be non-viral promoters (e.g., the human elongation factor-1α promoter) or viral promoters, such as cytomegalovirus (CMV) promoters, SV40 promoters, RSV promoters, and promoters found in long terminal repeats of mouse stem cell viruses.

[0096] The recombinant expression vectors of the present invention can be designed for transient expression, stable expression, or both. Furthermore, recombinant expression vectors can be prepared for constitutive expression or for inducible expression.

[0097] Furthermore, recombinant expression vectors can be prepared to contain suicide genes. As used herein, the term "suicide gene" refers to a gene that causes cell death in cells expressing a suicide gene. A suicide gene can be a gene that confers sensitivity to an agent (e.g., a drug) to cells expressing the gene, and causes cell death when the cell comes into contact with or is exposed to the agent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0098] Another aspect of the invention provides isolated or purified TCRs, polypeptides, or proteins encoded by any nucleic acid or vector described herein with respect to other aspects of the invention.

[0099] Another aspect of the invention provides isolated or purified TCRs, polypeptides, or proteins produced by the expression in cells of any nucleic acid or vector described herein with respect to other aspects of the invention.

[0100] Another aspect of the invention provides a host cell comprising any nucleic acid or recombinant expression vector described herein. As used herein, the term "host cell" refers to any type of cell that may comprise the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell (e.g., plant, animal, fungus, or algae) or a prokaryotic cell (e.g., bacteria or protists). The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism (e.g., human)). The host cell may be an adherent cell or a suspension cell (i.e., a cell growing in suspension). Suitable host cells are known in the art, including, for example, *Escherichia coli* DH5α (… E. coliThe host cells include DH5α cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. For the purpose of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, such as DH5α cells. For the purpose of producing recombinant TCRs, polypeptides, or proteins, the host cell is preferably a mammalian cell. More preferably, the host cell is a human cell. For example, the host cell can be a human lymphocyte. In one aspect of the invention, the host cell is selected from T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, natural killer (NK) cells, macrophages, pluripotent cells, and specialized cells. Although the host cell can be any cell type, can originate from any type of tissue, and can be at any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell.

[0101] For the purposes of this document, T cells can be any type of T cell, such as cultured T cells (e.g., primary T cells), or T cells derived from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. Preferably, the T cells are human T cells. T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4+. + / CD8 + Double-positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD4 + T cells, CD8 + T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc.

[0102] In one aspect of the invention, the host cell is a pluripotent cell or a multipotent cell. A pluripotent cell has the ability to produce any one of the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent cells may include, for example, stem cells, such as embryonic stem cells, embryonic stem cells derived from nuclear transfer, induced pluripotent stem cells (iPSCs), etc. Multipotent cells may include, for example, hematopoietic stem cells. Modifying (e.g., reprogramming) a cell to a pluripotent state refers to reversing a cell into a pluripotent cell, and is described in, for example, Crompton et al. Trends ImmunolIn ., 35(4): 178-185 (2014). Exemplary techniques may include somatic cell nuclear transfer (SCNT), cell-cell fusion, and directed reprogramming. Examples of methods for implementing cell-cell fusion are described, for example, in Ogle et al., Nat. Rev. Mol. Cell Biol 6: 567-75 (2005) and Zhou et al., Cell Stem Cell In, 3: 382-388 (2008). Examples of methods for implementing SCNT are described, for example, in Hanna et al., Cell , 143: 508-525 (2010); Stadtfeld et al., Genes Dev ., 24: 2239-2263 (2010); Wilmut et al., Nature , 385: 810-813(1997); Vizcardo et al., Cell Stem Cell , 12: 31-36 (2013); and Crompton et al., Cell Stem Cell In , 12: 6-8 (2013). In one aspect of the invention, the host cell is an iPSC prepared by reprogramming any host cell described herein (e.g., T cell, NK cell, or constant natural killer T cell) into a pluripotent state.

[0103] One aspect of the invention also provides a cell population comprising at least one host cell described herein. The cell population can be a heterogeneous population comprising host cells that, in addition to at least one other cell type (e.g., host cells (e.g., T cells) or cells other than T cells (e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, myocytes, brain cells, etc.) that also contain any of the recombinant expression vectors described herein. Optionally, the cell population can be a substantially homogeneous population, wherein the population primarily comprises host cells containing recombinant expression vectors (e.g., substantially composed of host cells containing recombinant expression vectors). The population can also be a clonal cell population, wherein all cells in the population are clones of a single host cell containing a recombinant expression vector, such that all cells in the population contain the recombinant expression vector. In one aspect of the invention, the cell population is a clonal population comprising host cells containing the recombinant expression vectors described herein.

[0104] In one aspect of the invention, the number of cells in the population can be rapidly increased. The increase in the number of T cells can be achieved by any of a variety of methods known in the art, for example, as described in U.S. Patent Nos. 8,034,334; 8,383,099; and 11,401,503; Dudley et al. J. Immunother. , 26:332-42(2003); and Riddell et al., J. Immunol. Methods In , 128:189-201(1990). On the one hand, the expansion of T cell numbers is carried out by culturing T cells in the presence of OKT3 antibody, IL-2 and fed PBMCs (e.g., irradiated allogeneic PBMCs).

[0105] One aspect of the present invention provides a method for producing any TCR, polypeptide, or protein described herein, the method comprising culturing any host cell or host cell population described herein to produce a TCR, polypeptide, or protein.

[0106] Another aspect of the present invention is to generate an expression of human p53 Y220C or person p53 R175H A method for host cells having an antigen-specific TCR with an amino acid sequence, the method comprising contacting cells with any of the recombinant expression vectors of the present invention described herein, provided that a vector is allowed to be introduced into the cells.

[0107] Another aspect of the invention provides a method for generating engineered human cells (or populations of engineered human cells), the method comprising introducing any of the nucleic acid or recombinant expression vectors of the invention described herein into isolated human cells (or populations of isolated human cells), wherein the nucleic acid or recombinant expression vector comprises a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins of the invention described herein. The isolated human cells or populations of isolated human cells into which the nucleic acid or recombinant expression vector is introduced may be as described herein with respect to other aspects of the invention.

[0108] The TCRs, peptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention can be isolated and / or purified. As used herein, the term "isolation" means removal from their natural environment. As used herein, the term "purification" means an increase in purity, wherein "purification" is a relative term and is not necessarily interpreted as absolute purity. For example, purity may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or may be about 100%.

[0109] The TCRs, peptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention, all collectively referred to below as "TCR materials of the present invention," can be formulated into compositions, such as pharmaceutical compositions. In this regard, one aspect of the present invention provides pharmaceutical compositions comprising any TCR, peptide, protein, nucleic acid, expression vector, and host cell (including populations thereof) described herein, as well as pharmaceutically acceptable vectors. Pharmaceutical compositions of the present invention comprising any TCR material of the present invention may comprise more than one TCR material of the present invention, such as peptides and nucleic acids, or two or more different TCRs. Optionally, the pharmaceutical composition may comprise a combination of the TCR material of the present invention with other pharmaceutical activators or drugs, such as chemotherapeutic agents (e.g., asparaginase, busulfan, carboplatin, cisplatin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, etc.).

[0110] Preferably, the carrier is a pharmaceutically acceptable carrier. Regarding the pharmaceutical composition, the carrier can be any of those carriers conventionally used for the specific TCR material of the present invention. Methods for preparing the administerable composition are known or obvious to those skilled in the art and are described in more detail, for example, Remington: The Science and Practice of Pharmacy In the 23rd edition, Academic Press (2020). Preferably, a pharmaceutically acceptable carrier is one that does not have harmful side effects or toxicity under the conditions of use.

[0111] The choice of carrier will be determined in part by the specific TCR material of the present invention and the specific method of administering the TCR material of the present invention. Therefore, various suitable formulations of the pharmaceutical compositions of the present invention exist. Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intra-arterial, intrathecal, intratumoral, intratumoral, or intraperitoneal administration. More than one route may be used to administer the TCR material of the present invention, and in some cases, a particular route may provide a more direct and effective response than others.

[0112] Preferably, the TCR material of the present invention is administered by injection, for example, intravenously. When the TCR material of the present invention is a host cell expressing the TCR of the present invention, the pharmaceutically acceptable carrier for the injected cells may include any isotonic carrier, such as physiological saline (about 0.90% w / v aqueous NaCl solution, about 300 mOsm / L aqueous NaCl solution, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASM-LYTE A (Baxter, Deerfield, IL), about 5% aqueous glucose solution, or Ringer's lactate. Alternatively, the pharmaceutically acceptable carrier may be supplemented with human serum albumin.

[0113] The amount or dose of the TCR material of the present invention applied (e.g., the number of cells when the TCR material of the present invention is one or more cells) should be sufficient to induce, for example, a therapeutic or preventative response in a subject or animal within a reasonable timeframe. For example, the dose of the TCR material of the present invention should be sufficient to bind to a cancer antigen (e.g., mutated p53) or to detect, treat, or prevent cancer for a period of about 2 hours or longer (e.g., 12 to 24 hours or longer) from the time of application. In some respects, this timeframe may even be longer. The dose will be determined based on the efficacy of the specific TCR material of the present invention and the condition of the animal (e.g., a human) and the weight of the animal (e.g., a human) to be treated.

[0114] Many assays for determining the dosage are known in the art. For example, one assay may be used to determine the initial dose to be administered to a mammal, said assay comprising, in mammals in a group each given a different dose of T cells, comparing the extent of target cell lysis or IFN-γ secretion by T cells expressing the TCR, polypeptide, or protein of the present invention after administration of a given dose of such T cells to the mammal. The extent of target cell lysis or IFN-γ secretion after administration of a given dose can be determined by methods known in the art.

[0115] The dosage of the TCR material of the present invention will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular TCR material of the present invention. Typically, the attending physician will consider a variety of factors (e.g., age, weight, general health, diet, sex, the TCR material of the present invention to be administered, the route of administration, and the severity of the cancer being treated) to determine the dosage of the TCR material of the present invention for each individual patient. Regarding the aspect where the TCR material of the present invention is a cell population, the number of cells administered per infusion can vary, for example, approximately 1 x 102 6 Approximately 2x10 6 One or more. In some respects, less than 1x10 can be applied.6 Each cell.

[0116] Those skilled in the art will readily understand that the TCR material of the present invention can be modified in various ways to enhance its therapeutic or preventative efficacy. For example, the TCR material of the present invention can be conjugated directly or indirectly to a chemotherapeutic agent via a bridging agent. The practice of conjugating compounds to chemotherapeutic agents is known in the art. Those skilled in the art will recognize that sites on the TCR material of the present invention that are not functionally essential are ideal sites for connecting bridging agents and / or chemotherapeutic agents, provided that the bridging agents and / or chemotherapeutic agents, once connected to the TCR material of the present invention, do not interfere with the function of the TCR material of the present invention, i.e., its ability to bind mutated p53 or detect, treat, or prevent cancer.

[0117] The pharmaceutical compositions, TCRs, peptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations of the present invention are contemplated for use in methods of treating or preventing cancer. Without being limited to any particular theory, it is believed that the TCRs of the present invention specifically bind to mutated p53, such that the TCR (or the associated peptide or protein of the present invention), when expressed by cells, can mediate an immune response against target cells expressing mutated p53. In this regard, one aspect of the present invention provides a method of treating or preventing cancer in mammals, comprising administering to a mammal any of the pharmaceutical compositions, TCRs, peptides, or proteins described herein in an amount effective for treating or preventing cancer in mammals, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, peptides, or proteins described herein, or any host cell or cell population comprising a recombinant vector encoding any of the TCRs, peptides, or proteins described herein.

[0118] One aspect of the invention provides any pharmaceutical composition, TCR, polypeptide, or protein described herein for use in the treatment or prevention of cancer in mammals, comprising any nucleic acid or recombinant expression vector encoding a nucleotide sequence of any TCR, polypeptide, or protein described herein, or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide, or protein described herein.

[0119] As used herein, the terms “treatment” and “prevention”, and words derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, different degrees of treatment or prevention exist, which those skilled in the art consider to have potential benefit or therapeutic effect. In this regard, the methods of the present invention can provide treatment or prevention of cancer in any amount or level in mammals. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of cancer that are being treated or prevented. For example, treatment or prevention can include promoting tumor regression. Additionally, for the purposes of this document, “prevention” can include delaying the onset of cancer, or its symptoms or conditions. Optionally or additionally, “prevention” can include preventing or delaying the recurrence of cancer, or its symptoms or conditions.

[0120] It is also anticipated that the pharmaceutical compositions, TCRs, peptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations of the present invention can be used in methods for inducing an immune response against cancer in mammals. In this regard, one aspect of the invention provides a method for inducing an immune response against cancer in mammals, comprising administering to a mammal any of the pharmaceutical compositions, TCRs, peptides, or proteins described herein, in an amount effective in inducing an immune response against cancer in mammals, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, peptides, or proteins described herein, or any host cell or cell population comprising a recombinant vector encoding any of the TCRs, peptides, or proteins described herein.

[0121] One aspect of the invention provides for use in inducing an immune response against cancer in mammals, any pharmaceutical composition, TCR, polypeptide, or protein described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any TCR, polypeptide, or protein described herein, or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide, or protein described herein.

[0122] Another aspect of the invention provides a method for detecting the presence of cancer in mammals. The method comprises (i) contacting a sample containing one or more cells from a mammal with any of the TCRs, peptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or pharmaceutical compositions of the present invention as described herein, thereby forming a complex, and (ii) detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal.

[0123] The method of the present invention for detecting cancer in mammals may be a sample containing whole cells, their lysates, or fractions of whole cell lysates (e.g., nuclear or cytoplasmic fractions, whole protein fractions, or nucleic acid fractions).

[0124] For the purposes of the detection method of the present invention, the contact can be performed in vitro or in vivo with respect to mammals. Preferably, the contact is in vitro.

[0125] Furthermore, the detection of the complex can be performed by any number of methods known in the art. For example, the TCRs, peptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations of the present invention described herein can be labeled with detectable markers, such as, for example, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0126] For the purposes of the method of the present invention, when a host cell or cell population is applied, the cell may be a mammalian allogeneic or autologous cell. Preferably, the cell is an autologous mammalian cell.

[0127] Regarding the method of this invention, the cancer can be any cancer, including, for example, any acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer. Cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer. In a preferred aspect, the cancer is a cancer expressing a mutated p53. The cancer may express a p53 with a mutation at one or both of positions 220 and 175 as defined in SEQ ID NO: 1. The cancer may express a p53 with one or two of the following human p53 mutations: Y220C and R175H. In one aspect of the invention, the cancer is epithelial cancer. In another aspect of the invention, the cancer is cholangiocarcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer (NSCLC), glioblastoma, cervical cancer, head and neck cancer, breast cancer, pancreatic cancer, or bladder cancer. Cancers are known to contain the Y220C or R175H mutation in human p53.

[0128] The mammals mentioned in the methods of this invention can be any mammal. As used herein, the term "mammal" means any mammal, including but not limited to rodents (e.g., mice and hamsters) and lagomorphs (e.g., rabbits). Preferably, the mammals are from the order Carnivora, including felines (cats) and canines (dogs). More preferably, the mammals are from the order Artiodactyla, including bovids (cows) and suidae (pigs) or perissodactyls, including equines (horses). Most preferably, the mammals are from the orders Primates, Cebomorphs, or Simoids (monkeys) or Anthropoids (humans and apes). A particularly preferred mammal is the human.

[0129] The following embodiments further illustrate the invention, but should not be construed as limiting its scope in any way.

[0130] Example 1

[0131] This example illustrates the isolation of TCR from patient 4196.

[0132] p53 R175H Epitopes of 9 amino acids (HMTEVVR) H C)(SEQ ID NO: 112) contains a cysteine ​​residue at its C-terminus. Under oxidative conditions, the cysteine ​​residue can be oxidized and form disulfide bonds with other cysteine ​​residues, potentially interfering with its ability to bind to MHC molecules (Sachs et al., J. Immunol ., 205(2): 539–549(2020)). This can lead to a decrease in T cell activity. To minimize this effect, in the experiments described in the following examples, at (HMTEVVR) H The native cysteine ​​at position 9 of C (SEQ ID NO: 112) is replaced with α-aminobutyric acid (AABA) to produce p53. R175H HMTEVVR (a peptide analog) H X (SEQ ID NO: 116), where X at position 9 of SEQ ID NO: 116 is AABA. AABA is a cysteine ​​analog containing a thiol group present in methyl-substituted cysteine. (HMTEVVR) R The native cysteine ​​residue at position 9 of C)(SEQ ID NO:113) is replaced with AABA to produce the corresponding wild-type mimic peptide HMTEVVR. R X(SEQ ID NO: 117), where X at position 9 of SEQ ID NO: 117 is AABA.

[0133] p53 was isolated from TILs (4196 TILs) from patient 4196. R175H Prior to the specific TCR, three different culture conditions were tested.

[0134] Tetramer staining of 4196 TILs was performed for p53. R175H Detection of reactive cells. Following two weeks of culture as outlined in Table 1, HLA-A2 tetramers were stained in T cells. HLA-A2 tetramers are cells loaded with p53... R175H HMTEVVR, an epitope analog of 9 amino acids H The oligomerization of HLA-A2 monomers of X (SEQ ID NO: 116) (where X at position 9 of SEQ ID NO: 116 is AABA). The frequency of tetramer-positive cells observed for each culture condition 1, 2, and 3 is shown in Figure 1A middle.

[0135] Table 1

[0136]

[0137] From the display of specific identification using p53 R175H Three TCRs were isolated from tumor-infiltrating lymphocytes (TILs) of antigen-presenting cells (APCs) pulsed with peptide analogs instead of the corresponding wild-type (WT) peptide analogs. Subsequently, fluorescence-activated cell sorting (FACS) was performed based on the upregulation of T cell activation markers 4-1BB and OX-40. The three TCRs were 4196-C TCR (TRAV38-1 / TRBV5-1), 4196-D TCR (TRAV14 / TRBV7-9), and 4196-E TCR (TRAV10 / TRBV6-6).

[0138] The sequences of the variable regions of the TCR α and β chains were identified using single-cell TCR sequencing. The amino acid sequences of the variable regions of the α and β chains are shown in Table 2. Underlined terms are CDRs. Bold terms are N-terminal signal peptides.

[0139] Table 2

[0140]

[0141] Example 2

[0142] This example illustrates the isolation of TCR from patient 4424.

[0143] TILs from patient 4424 were stained for tetramer for p53. R175HDetection of reactive cells. Following two weeks of culture as outlined in Table 1, HLA-A2 tetramers were stained in T cells. HLA-A2 tetramers are cells loaded with p53... R175H HMTEVVR, an epitope analog of 9 amino acids H The oligomerized HLA-A2 monomer of X (SEQ ID NO: 116) (where X at position 9 of SEQ ID NO: 116 is AABA). Flow cytometry staining of CD8 and tetramers showed... Figure 2A In the middle, 4424-R175HTCR separated from the tetramer. Because the tetramer is constructed from HLA-A2 monomers, these staining data indicate that the 4424-R175H TCR will be HLA-A2 restricted. Although other TCRs targeting the mutation p53 have been previously identified via co-culture experiments, 4424-R175HTCR (TRAV10 / TRBV2) is the first TCR identified by tetramerization.

[0144] The sequences of the variable regions of the TCR α and β chains were identified using single-cell TCR sequencing. The amino acid sequences of the variable regions of the α and β chains are shown in Table 3. Underlined terms represent CDRs. Bold terms represent N-terminal signal peptides.

[0145] Table 3

[0146]

[0147] Example 3

[0148] This example illustrates the isolation of TCR from patient 4402.

[0149] TILs from patient 4402 showed specific recognition using p53. Y220C Peptide (DRNTFRHSVVVP) C Antigen-presenting cells (APCs) were pulsed with EPPEVGSDCTTI (SEQ ID NO: 114) instead of the corresponding wild-type (WT) peptide (DRNTFRHSVVVPYEPPEVGSDCTTI) (SEQ ID NO: 115). Subsequently, FACS was performed based on the upregulation of T cell activation markers 4-1BB and OX-40. Twenty-one (21) TCRs (4402-Y220C-A to -U, respectively) were isolated from TILs from patient 4402.

[0150] After co-culturing with healthy donor PBLs expressing the corresponding 21 TCRs isolated from TILs (effective cells) of patient 4402, ELISpot analysis was performed to detect IFN-γ release. The release was compared to the DMSO control with IFN-γ released at 1 µg / mL of the mutated p53 Y220C peptide (DRNTFRHSVVVP).C B cells (target cells) pulsed with DMSO (4402-Y220C-B TCR, 4402-Y220C-C TCR, and 4402-Y220C-L TCR showed increased IFN-γ release, while the other 18 TCRs did not. Effector cells treated with phorbol 12-myristate 13-acetate (PMA) and iomycin served as positive controls. Controls included: cultures containing only effector cells, cultures containing target cells pulsed with DMSO, and cultures containing PBL transduced with an empty vector (simulated).

[0151] The sequences of the variable regions of the α and β chains of the TCRs were identified using single-cell TCR sequencing. The amino acid sequences of the variable regions of the α and β chains of three TCRs (4402-Y220C-BTCR (TRAV12-3 / TRBV7-2), 4402-Y220C-C TCR (TRAV26-2 / TRBV5-6), and 4402-Y220C-L TCR (TRAV23 / TRBV5-1)) are shown in Table 4. Underlined CDRs are used. Bold text indicates the N-terminal signal peptide.

[0152] Table 4

[0153]

[0154] Example 4

[0155] This embodiment illustrates the construction of retroviral vectors encoding the respective TCRs of Examples 1 to 3.

[0156] Nucleotide sequences of the variable regions of the α and β chains of the TCRs encoding Tables 2 to 4 were obtained and codon optimization was performed. The TCRβ VDJ region was fused to the mouse TCRβ constant chain. The TCRα VJ region was fused to the mouse TCRα constant chain. Regardless of specific theories or mechanisms, it is believed that replacing the constant regions of the human TCRα and TCRβ chains with the corresponding mouse constant regions improves TCR expression and function (Cohen et al.). Cancer Res., 66(17): 8878-86(2006)).

[0157] Furthermore, the mouse TCRα and TCRβ constant chains are cysteine ​​modified. A transmembrane hydrophobic mutation was introduced into the mouse TCRα constant chain. Without being limited to any specific theory or mechanism, it is believed that these modifications lead to preferential pairing of the introduced TCR chains and enhanced TCR surface expression and function. (Cohen et al.) Cancer Res., 67(8):3898-903(2007); Haga-Friedman et al., J. Immu., 188: 5538–5546 (2012)).

[0158] The full-length α and β chains of each of the seven TCRs, including these modifications for the constant regions, are shown in Table 5. In Table 5, underlined CDRs are CDRs, italics are constant regions, and underlined and bolded amino acid residues are modified in the constant regions.

[0159] Table 5

[0160]

[0161] For each TCR in Table 5, the nucleotide sequences encoding the α and β chains were cloned into an MSGV1-based retroviral vector. The TCRβ and TCRα chains were separated by the furin Ser / Gly P2A linker RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 103). Without being limited to any specific theory or mechanism, it is assumed that the linker provides comparable expression efficiency for both chains (Szymczak et al., Nat. Biotechnol., 22(5):589-94(2004)).

[0162] Example 5

[0163] This example illustrates the specificity and affinity of 4196-C TCR, 4196-D TCR, and 4196-E TCR.

[0164] Healthy donor PBLs were transduced using retroviral vectors encoding 4196-C TCR, 4196-D TCR, or 4196-E TCR as described in Example 4, or TCRs 4196_AV6_ and _BV11-2 disclosed in US 2020 / 0277352. T cells expressing the TCRs were co-cultured with T2 cells expressing HLA-A2 and already conjugated with the wild-type epitope analog HMTEVVRRX (SEQ ID NO: 117), wherein the X at position 9 of SEQ ID NO: 117 is AABA; or with p53 R175H Epitope analogs of 9 amino acids in Figures 1B to 1EIncubation was performed at the concentrations shown. Following co-culture, the T cell activation marker 4-1BB was measured by flow cytometry. The 4196-C, 4196-D, and 4196-E TCRs all showed specificity for the mutant p53 epitope analogue. Conversely, TCRs 4196_AV6_ and _BV11-2 showed significant recognition of the wild-type epitope analogue at concentrations of 1000 ng / mL and 100 ng / mL, respectively. These results indicate that the 4196-CTCR, 4196-D, and 4196-E TCRs exhibit higher specificity for the p53 R175H epitope analogue compared to TCRs 4196_AV6_ and _BV11-2.

[0165] Redraw Figures 1B to 1E The titration curve is used for comparison, such as... Figure 1F As shown. Compared to TCRs 4196_AV6_ and _BV11-2, the 4196-C TCR, 4196-D TCR, and 4196-E TCR exhibited better (4196-C TCR and 4196-E TCR) or equivalent (4196-D TCR) affinity for the mutated p53 R175H epitope analogues. These results suggest that the 4196-C TCR, 4196-D TCR, and 4196-E TCR potentially possess higher sensitivity to the p53 R175H antigen compared to TCRs 4196_AV6_ and _BV11-2.

[0166] Example 6

[0167] This example illustrates how 4196-C TCR, 4196-D TCR, and 4196-E TCR kill tumor cells.

[0168] Effector cells were prepared by transducing healthy donor PBLs with retroviral vectors encoding 4196-C TCR, 4196-D TCR, or 4196-E TCR as described in Example 4. The antitumor activity of the 4196-C TCR, 4196-D TCR, or 4196-E TCR was determined using the INCUCYTE immunocytotoxicity assay. TYK-nu human ovarian cancer cells (target cells) naturally expressing p53 R175H and HLA-2 were labeled with red fluorescent protein (RFP). 50,000 effector cells were co-cultured with 10,000 TYK-nu cells over 3 days. The number of viable RFP+TYK-nu cells was counted at 4-hour intervals. Results were shown... Figure 1G These results demonstrate that all 4196-C TCR, 4196-D TCR, or 4196-E TCR can mediate significant tumor cell killing in vitro.

[0169] Example 7

[0170] This example illustrates the specificity and affinity of 4424-R175H TCR.

[0171] Effector cells were healthy donor PBLs transduced with the retroviral vector encoding 4424-R175H TCR as described in Example 4. Target cells were monkey kidney COS7 cells engineered to express HLA-A2 and conjugated with the wild-type epitope analog HMTEVVRRX (SEQ ID NO: 117), wherein the X at position 9 of SEQ ID NO: 117 is AABA; or conjugated with mutated p53. R175H peptide analogue HMTEVVR H X (SEQ ID NO: 116), where X at position 9 of SEQ ID NO: 116 is AABA, in Figure 2B Incubation was performed at the concentrations shown. After 18 hours of co-culture, the T cell activation marker 4-1BB was measured by flow cytometry. Results were shown... Figure 2B The data showed that the 4424-R175H TCR is specific for the mutated p53 R175H. Since the COS7 cells used here only express HLA-A2, these data confirm the HLA restriction of the 4424-R175H TCR by HLA-A2.

[0172] Example 8

[0173] This example illustrates a functional comparison of various TCRs that respond to p53 R175H.

[0174] The INCUCYTE immunocytotoxic assay, as described in Example 6, was used to compare the ability of various TCRs targeting p53 R175H to kill tumor cells in vitro. TCR 4141-TCR1a2, disclosed in US 2020 / 0332785, was included as a reference. 20,000 healthy donor T cells (effector cells) expressing 4196-C, 4196-D, 4196-E, or 4424-R175H TCRs encoded by their respective retroviral vectors as described in Example 4, or expressing TCR 4141-TCR1a2, were co-cultured with 10,000 human ovarian cancer TYK-nu cells (target cells) at a 2:1 effector-to-target ratio for 72 hours. Live RFP+TYK-nu cells were counted every 3 hours. Results were shown... Figure 3 In contrast to the tumor-only condition, all T-cell-treated conditions showed a significant delay in tumor growth. Compared to the 4141-TCR1a2 TCR, the 4196-C TCR, 4196-DTCR, and 4424-R175H TCR showed a significant reduction in tumor growth.

[0175] Example 9

[0176] This embodiment illustrates the identification of tumor cells using 4402-Y220C-B TCR, 4402-Y220C-C TCR, and 4402-Y220C-L TCR.

[0177] Healthy donor PBLs (effect cells) were independently transduced using the respective retroviruses of Example 4 encoding 4402-Y220C-B TCR, 4402-Y220C-C TCR, or 4402-Y220C-L TCR. Effector cells and target cells were co-cultured under the conditions shown in Table 6.

[0178] Table 6

[0179]

[0180] IFN-γ release was measured using the ELISpot assay. Unrelated organoid tumor cells from different patients were included as negative controls. Autologous B cells pulsed with p53 Y220C 25-mer (DRNTFRHSVVVPCEPPEVGSDCTTI) (SEQ ID NO:114) were included as positive controls.

[0181] When autologous organoid tumor cells are used with p53 Y220C 25-mer (DRNTFRHSVVVP) C When pulsed with p53 Y220C 25-mer (DRNTFRHSVVVP), the 4402-Y220C-C TCR and 4402-Y220C-L TCR release IFN-γ. These results indicate that tumor cells express HLA molecules that restrict the release of IFN-γ from the 4402-Y220C-C TCR and 4402-Y220C-L TCR. C During the pulse of EPPEVGSDCTTI (SEQ ID NO: 114), the 4402-Y220C-B TCR does not release IFN-γ. Autologous organoid tumor cells exhibit a loss of HLA heterozygosity, and they may have lost HLA-DRB3, which restricts the 4402-Y220C-B TCR. 02:02 molecules.

[0182] Example 10

[0183] This example illustrates the affinity and specificity of 4402-Y220C-B TCR, 4402-Y220C-C TCR, and 4402-Y220C-L TCR.

[0184] Healthy donor PBLs (effective cells) were transduced using the retroviral vector encoding 4402-Y220C-B, 4402-Y220C-C, or 4402-Y220C-L TCRs as described in Example 4, or the retroviral vector encoding 4343-D TCRs disclosed in US 2023 / 0321240. Target cells were prepared with WT (DRNTFRHSVVVP). Y EPPEVGSDCTTI (SEQ ID NO: 115) or the mutant (DRNTFRHSVVVP) C EPPEVGSDCTTI)(SEQ ID NO: 114) peptide in Figures 4A to 4D Autologous B cells pulsed at the concentration shown. After co-culturing effector cells and target cells, 4-1BB was measured by flow cytometry. Results showed... Figures 4A to 4D In the study, the 4402-Y220C-B, 4402-Y220C-C, and 4402-Y220C-L TCRs showed minimal WT p53 peptide recognition. Conversely, the 4343-D TCR showed significant WT p53 peptide recognition. Compared to the 4343-D TCR, the 4402-Y220C-BTCR, 4402-Y220C-C, and 4402-Y220C-L TCRs exhibited better mutant specificity.

[0185] Example 11

[0186] This example illustrates that 4402-Y220C-B TCR is HLA-DRB3. 02:02 is restricted.

[0187] Healthy donor PBLs (effective cells) were transduced using the retroviral vector encoding the 4402-Y220C-B TCR from Example 4. Target cells were COS7 cells independently transfected with a single class II HLA molecule expressed by the patient (Table 7).

[0188] Table 7

[0189]

[0190] Effector cells and target cells were co-cultured. After co-culture, IFN-γ secretion was measured using ELISApot. When HLA-DRB3 was expressed... When COS7 cells were co-cultured at 02:02, cells expressing 4402-Y220C-B TCR showed significant IFN-γ secretion.

[0191] Example 12

[0192] This embodiment illustrates the recognition of different autologous tumor organoids by the 4402-Y220C-C TCR and the 4402-Y220C-L TCR.

[0193] Healthy donor PBLs (effect cells) were transduced using the retroviral vector encoding 4402-Y220C-C TCR or 4402-Y220C-L TCR as described in Example 4. Target cells were autologous breast tumor organoids from passage 11 or 27 of the patient's 4402 cell line. Target cells pulsed with DMSO were used as controls.

[0194] Effector cells and target cells were co-cultured. T cell activation was measured by upregulation of 4-1BB. Results showed... Figure 4E The results showed that both TCRs recognized only the 27th generation organoid cells, suggesting that the 11th generation organoid cells may have lost the HLA molecules that restrict the 4402-Y220C-C TCR and the 4402-Y220C-L TCR.

[0195] Example 13

[0196] This example illustrates the HLA-DRB1 of the 11th and 27th generation autologous organoids from Example 12. 15:01 and DRB1 Differential expression of molecules at 13:03.

[0197] HLA-DRB1 of the 11th and 27th generation autologous organoid cells in Example 12 15:01 and DRB1 Expression at 13:03 was measured using transcriptome analysis (RNA sequencing). Results showed... Figure 4F The Transcripts Per Kilobase Million (TPM) values ​​of the two HLA molecules support HLA-DRB1 in 11th generation organoid cells. The loss of 13:0. These results indicate the presence of HLA-DRB1. 13:0 is the limiting element for 4402-Y220C-C TCR and 4402-Y220C-L TCR.

[0198] Example 14

[0199] This example illustrates that adoptive transfer of cells transduced with the retroviral vector encoding 4196-C TCR of Example 4 reduced tumor volume in tumor-bearing mice.

[0200] NSG mice (immunely compromised) were subcutaneously injected with naturally expressed p53 R175H mutation and HLA-A. TYK-nu human ovarian cancer cells at 02:01. Two weeks later, tumor-bearing mice were randomized and subjected to adoptive cell transfer using independently transduced healthy donor T cells:

[0201] 1. The retroviral vector encoding the 4196-C TCR of Example 4;

[0202] 2. Unrelated TCR control (i.e., 4259);

[0203] 3. TCR 4196_AV12-1_ and _BV6-1 disclosed in US 2020 / 0277352;

[0204] 4. TCR 4196_AV38-1_ and _BV10-3 disclosed in US 2020 / 0277352;

[0205] 5. TCR 4196_AV6_ and _BV11-2 disclosed in US 2020 / 0277352;

[0206] 6. TCR 4141-TCR1a2 disclosed in US 2020 / 0332785; or

[0207] 7. 4141 IVS TCR disclosed in U.S. Patent Application No. 18 / 289,596.

[0208] Two T-cell dose levels were tested: 2 million or 10 million T cells / mouse. Mean tumor size was measured at different time points over a period of up to 40 days following adoptive transfer of the transduced cells.

[0209] Results obtained from a dose of 2 million T cells / mouse showed Figure 6 In the middle. For example Figure 6 As shown, only the 4196-C TCR showed statistical significance compared to the 4259 TCR and the unrelated TCR control.

[0210] Results obtained from a dose of 10 million T cells / mouse showed Figure 7 In the middle. For example Figure 7 As shown, only the 4196-C TCR and TCRs 4196_AV6_ and _BV11-2 showed statistical significance relative to the 4259 TCR and the unrelated TCR control.

[0211] All references cited in this article, including publications, patent applications and patents, are incorporated herein by reference to the extent that each reference is individually and specifically indicated by reference and is listed in its entirety in this article.

[0212] Unless otherwise stated herein or obviously contradicted by the context, in the context of describing the invention (particularly in the context of the appended claims), the use of the terms “a,” “an,” “the,” and “at least one,” as well as similar referents, shall be interpreted as including both singular and plural forms. Unless otherwise stated herein or obviously contradicted by the context, the use of the term “at least one” followed by a list of one or more items (e.g., “at least one of A and at least one of B”) shall be interpreted as meaning one item selected from the list (A or B) or any combination of two or more of the list (A and B). Unless otherwise shown, the terms “comprising,” “having,” “including,” and “containing” shall be interpreted as open-ended terms (i.e., meaning “including but not limited to”). Unless otherwise stated herein, the description of ranges of values ​​herein is intended only as a shorthand method for individually referring to each individual value falling within that range, and each individual value is incorporated into this specification as if it were individually described herein. Unless otherwise stated herein or obviously contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all embodiments or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the invention and, unless otherwise stated, does not constitute a limitation on the scope of the invention. The language in this specification should not be construed as indicating that any unclaimed element is necessary for the implementation of the invention.

[0213] This document describes preferred aspects of the invention, including the best modes known to the inventors of this application for carrying out the invention. Variations of these preferred aspects will become apparent to those skilled in the art upon reading the foregoing description. The inventors of this application expect those skilled in the art to appropriately employ such variations, and the inventors intend to practice the invention in ways different from those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter described in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, the invention includes any combination of the foregoing elements in all possible variations.

Claims

1. Regarding people (p. 53) Y220C or person p53 R175H An isolated or purified T-cell receptor (TCR) with an antigen-specific amino acid sequence, wherein the TCR comprises the following amino acid sequence: (1) All SEQ ID NO: 2-7; (2) All SEQ ID NO: 16-21; (3) All SEQ ID NO: 30-35; (4) All SEQ ID NOs: 44-49; (5) All SEQ ID NO: 58-63; (6) All SEQ ID NO: 72-77; or (7) All SEQ ID NO: 86-91.

2. The TCR of claim 1, wherein the TCR comprises the following amino acid sequence: (1) SEQ ID NO: 8; (2) SEQ ID NO: 9; (3) SEQ ID NO: 8 and 9; (4) SEQ ID NO: 10; (5) SEQ ID NO: 11; (6) SEQ ID NO: 10 and 11; (7) SEQ ID NO: 22; (8) SEQ ID NO: 23; (9) SEQ ID NO: 22 and 23; (10) SEQ ID NO: 24; (11) SEQ ID NO: 25; (12) SEQ ID NO: 24 and 25; (13) SEQ ID NO: 36; (14) SEQ ID NO: 37; (15) SEQ ID NO: 36 and 37; (16) SEQ ID NO: 38; (17) SEQ ID NO: 39; (18) SEQ ID NO: 38 and 39; (19) SEQ ID NO: 50; (20) SEQ ID NO: 51; (21) SEQ ID NO: 50 and 51; (22) SEQ ID NO: 52; (23) SEQ ID NO: 53; (24) SEQ ID NO: 52 and 53; (25) SEQ ID NO: 64; (26) SEQ ID NO: 65; (27) SEQ ID NO: 64 and 65; (28) SEQ ID NO: 66; (29) SEQ ID NO: 67; (30) SEQ ID NO: 66 and 67; (31) SEQ ID NO: 78; (32) SEQ ID NO: 79; (33) SEQ ID NO: 78 and 79; (34) SEQ ID NO: 80; (35) SEQ ID NO: 81; (36) SEQ ID NO: 80 and 81; (37) SEQ ID NO: 92; (38) SEQ ID NO: 93; (39) SEQ ID NO: 92 and 93; (40) SEQ ID NO: 94; (41) SEQ ID NO: 95; or (42) SEQ ID NO: 94 and 95.

3. The TCR of claim 1 or 2, wherein the TCR comprises the following amino acid sequence: (1) SEQ ID NO: 12; (2) SEQ ID NO: 13; (3) SEQ ID NO: 12 and 13; (4) SEQ ID NO: 14; (5) SEQ ID NO: 15; (6) SEQ ID NO: 14 and 15; (7) SEQ ID NO: 26; (8) SEQ ID NO: 27; (9) SEQ ID NO: 26 and 27; (10) SEQ ID NO: 28; (11) SEQ ID NO: 29; (12) SEQ ID NO: 28 and 29; (13) SEQ ID NO: 40; (14) SEQ ID NO: 41; (15) SEQ ID NO: 40 and 41; (16) SEQ ID NO: 42; (17) SEQ ID NO: 43; (18) SEQ ID NO: 42 and 43; (19) SEQ ID NO: 54; (20) SEQ ID NO: 55; (21) SEQ ID NO: 54 and 55; (22) SEQ ID NO: 56; (23) SEQ ID NO: 57; (24) SEQ ID NO: 56 and 57; (25) SEQ ID NO: 68; (26) SEQ ID NO: 69; (27) SEQ ID NO: 68 and 69; (28) SEQ ID NO: 70; (29) SEQ ID NO: 71; (30) SEQ ID NO: 70 and 71; (31) SEQ ID NO: 82; (32) SEQ ID NO: 83; (33) SEQ ID NO: 82 and 83; (34) SEQ ID NO: 84; (35) SEQ ID NO: 85; (36) SEQ ID NO: 84 and 85; (37) SEQ ID NO: 96; (38) SEQ ID NO: 97; (39) SEQ ID NO: 96 and 97; (40) SEQ ID NO: 98; (41) SEQ ID NO: 99; or (42)SEQ ID NO: 98 and 99.

4. The TCR as described in any one of claims 1 to 3, wherein the human p53 Y220C The amino acid sequence is DRNTFRHSVVVP C EPPEVGSDCTTI (SEQ ID NO: 114).

5. The TCR as claimed in any one of claims 1 to 4, wherein the TCR is effective against DRNTFRHSVVVP. Y The wild-type human p53 amino acid sequence of EPPEVGSDCTTI (SEQ ID NO: 115) is not antigen-specific.

6. The TCR as described in any one of claims 1 to 3, wherein the human p53 R175H The amino acid sequence is HMTEVVR H C(SEQ ID NO: 112).

7. The TCR as claimed in any one of claims 1 to 3 and 6, wherein the TCR is effective against HMTEVVR. R The wild-type human p53 amino acid sequence of C (SEQ ID NO: 113) does not have antigen specificity.

8. An isolated or purified polypeptide comprising the functional portion of the TCR according to any one of claims 1 to 7, wherein the polypeptide comprises the following amino acid sequence: (1) All SEQ ID NO: 2-7; (2) All SEQ ID NO: 16-21; (3) All SEQ ID NO: 30-35; (4) All SEQ ID NO: 44-49; (5) All SEQ ID NO: 58-63; (6) All SEQ ID NO: 72-77; or (7) All SEQ ID NO: 86-91.

9. The polypeptide of claim 8, wherein the polypeptide comprises the following amino acid sequence: (1) SEQ ID NO: 8; (2) SEQ ID NO: 9; (3) SEQ ID NO: 8 and 9; (4) SEQ ID NO: 10; (5) SEQ ID NO: 11; (6) SEQ ID NO: 10 and 11; (7) SEQ ID NO: 22; (8) SEQ ID NO: 23; (9) SEQ ID NO: 22 and 23; (10) SEQ ID NO: 24; (11) SEQ ID NO: 25; (12) SEQ ID NO: 24 and 25; (13) SEQ ID NO: 36; (14) SEQ ID NO: 37; (15) SEQ ID NO: 36 and 37; (16) SEQ ID NO: 38; (17) SEQ ID NO: 39; (18) SEQ ID NO: 38 and 39; (19) SEQ ID NO: 50; (20) SEQ ID NO: 51; (21) SEQ ID NO: 50 and 51; (22) SEQ ID NO: 52; (23) SEQ ID NO: 53; (24) SEQ ID NO: 52 and 53; (25) SEQ ID NO: 64; (26) SEQ ID NO: 65; (27) SEQ ID NO: 64 and 65; (28) SEQ ID NO: 66; (29) SEQ ID NO: 67; (30) SEQ ID NO: 66 and 67; (31) SEQ ID NO: 78; (32) SEQ ID NO: 79; (33) SEQ ID NO: 78 and 79; (34) SEQ ID NO: 80; (35) SEQ ID NO: 81; (36) SEQ ID NO: 80 and 81; (37) SEQ ID NO: 92; (38) SEQ ID NO: 93; (39) SEQ ID NO: 92 and 93; (40) SEQ ID NO: 94; (41) SEQ ID NO: 95; or (42) SEQ ID NO: 94 and 95.

10. The polypeptide of claim 8 or 9, wherein the polypeptide comprises the following amino acid sequence: (1) SEQ ID NO: 12; (2) SEQ ID NO: 13; (3) SEQ ID NO: 12 and 13; (4) SEQ ID NO: 14; (5) SEQ ID NO: 15; (6) SEQ ID NO: 14 and 15; (7) SEQ ID NO: 26; (8) SEQ ID NO: 27; (9) SEQ ID NO: 26 and 27; (10) SEQ ID NO: 28; (11) SEQ ID NO: 29; (12) SEQ ID NO: 28 and 29; (13) SEQ ID NO: 40; (14) SEQ ID NO: 41; (15) SEQ ID NO: 40 and 41; (16) SEQ ID NO: 42; (17) SEQ ID NO: 43; (18) SEQ ID NO: 42 and 43; (19) SEQ ID NO: 54; (20) SEQ ID NO: 55; (21) SEQ ID NO: 54 and 55; (22) SEQ ID NO: 56; (23) SEQ ID NO: 57; (24) SEQ ID NO: 56 and 57; (25) SEQ ID NO: 68; (26) SEQ ID NO: 69; (27) SEQ ID NO: 68 and 69; (28) SEQ ID NO: 70; (29) SEQ ID NO: 71; (30) SEQ ID NO: 70 and 71; (31) SEQ ID NO: 82; (32) SEQ ID NO: 83; (33) SEQ ID NO: 82 and 83; (34) SEQ ID NO: 84; (35) SEQ ID NO: 85; (36) SEQ ID NO: 84 and 85; (37) SEQ ID NO: 96; (38) SEQ ID NO: 97; (39) SEQ ID NO: 96 and 97; (40) SEQ ID NO: 98; (41) SEQ ID NO: 99; or (42)SEQ ID NO: 98 and 99.

11. An isolated or purified protein comprising a first polypeptide chain and a second polypeptide chain, wherein: (1) The first polypeptide chain contains the amino acid sequence of all SEQ ID NO: 2-4; (2) The second polypeptide chain contains the amino acid sequence of all SEQ ID NO: 5-7; (3) The first polypeptide chain contains all the amino acid sequences of SEQ ID NO: 2-4 and the second polypeptide chain contains all the amino acid sequences of SEQ ID NO: 5-7; (4) The first polypeptide chain contains the amino acid sequence of all SEQ ID NO: 16-18; (5) The second polypeptide chain contains the amino acid sequence of all SEQ ID NO: 19-21; (6) The first polypeptide chain contains all the amino acid sequences of SEQ ID NO: 16-18 and the second polypeptide chain contains all the amino acid sequences of SEQ ID NO: 19-21; (7) The first polypeptide chain contains the amino acid sequence of all SEQ ID NO: 30-32; (8) The second polypeptide chain contains the amino acid sequence of all SEQ ID NO: 33-35; (9) The first polypeptide chain contains all the amino acid sequences of SEQ ID NO: 30-32 and the second polypeptide chain contains all the amino acid sequences of SEQ ID NO: 33-35; (10) The first polypeptide chain contains the amino acid sequence of all SEQ ID NO: 44-46; (11) The second polypeptide chain contains the amino acid sequence of all SEQ ID NO: 47-49; (12) The first polypeptide chain contains all the amino acid sequences of SEQ ID NO: 44-46 and the second polypeptide chain contains all the amino acid sequences of SEQ ID NO: 47-49; (13) The first polypeptide chain contains the amino acid sequence of all SEQ ID NO: 58-60; (14) The second polypeptide chain contains the amino acid sequence of all SEQ ID NO: 61-63; (15) The first polypeptide chain contains all the amino acid sequences of SEQ ID NO: 58-60 and the second polypeptide chain contains all the amino acid sequences of SEQ ID NO: 61-63; (16) The first polypeptide chain contains the amino acid sequence of all SEQ ID NO: 72-74; (17) The second polypeptide chain contains the amino acid sequence of all SEQ ID NO: 75-77; (18) The first polypeptide chain contains all the amino acid sequences of SEQ ID NO: 72-74 and the second polypeptide chain contains all the amino acid sequences of SEQ ID NO: 75-77; (19) The first polypeptide chain contains the amino acid sequence of all SEQ ID NO: 86-88; (20) The second polypeptide chain contains the amino acid sequence of all SEQ ID NO: 89-91; or (21) The first polypeptide chain contains all the amino acid sequences of SEQ ID NO: 86-88 and the second polypeptide chain contains all the amino acid sequences of SEQ ID NO: 89-91.

12. The protein of claim 11, wherein... (1) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 8; (2) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 9; (3) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 8 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 9; (4) The first polypeptide chain contains the amino acid sequence of SEQ ID NO:10; (5) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 11; (6) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 10 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 11; (7) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 22; (8) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 23; (9) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 22 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 23; (10) The first polypeptide chain contains the amino acid sequence of SEQ ID NO:24; (11) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 25; (12) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 24 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 25; (13) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 36; (14) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 37; (15) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 36 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 37; (16) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 38; (17) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 39; (18) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 38 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 39; (19) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 50; (20) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 51; (21) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 50 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 51; (22) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 52; (23) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 53; (24) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 52 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 53; (25) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 64; (26) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 65; (27) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 64 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 65; (28) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 66; (29) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 67; (30) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 66 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 67; (31) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 78; (32) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 79; (33) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 78 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 79; (34) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 80; (35) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 81; (36) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 80 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 81; (37) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 92; (38) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 93; (39) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 92 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 93; (40) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 94; (41) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 95; or (42) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 94 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO:

95.

13. The protein of claim 11 or 12, wherein... (1) The first polypeptide chain contains the amino acid sequence of SEQ ID NO:12; (2) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 13; (3) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 12 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 13; (4) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 14; (5) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 15; (6) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 14 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 15; (7) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 26; (8) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 27; (9) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 26 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 27; (10) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 28; (11) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 29; (12) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 28 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 29; (13) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 40; (14) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 41; (15) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 40 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 41; (16) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 42; (17) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 43; (18) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 42 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 43; (19) The first polypeptide chain contains the amino acid sequence of SEQ ID NO:54; (20) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 55; (21) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 54 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 55; (22) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 56; (23) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 57; (24) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 56 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 57; (25) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 68; (26) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 69; (27) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 68 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 69; (28) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 70; (29) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 71; (30) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 70 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 71; (31) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 82; (32) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 83; (33) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 82 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 83; (34) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 84; (35) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 85; (36) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 84 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 85; (37) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 96; (38) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 97; (39) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 96 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 97; (40) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 98; (41) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 99; or (42) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 98 and the second polypeptide chain contains the amino acid sequence of SEQ ID NO:

99.

14. A bispecifically binding TCR fusion protein comprising (i) a TCR as claimed in any one of claims 1 to 7, a polypeptide as claimed in any one of claims 8 to 10, or a protein as claimed in any one of claims 11 to 13, and (ii) an anti-CD3 effector.

15. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR as described in any one of claims 1 to 7, a polypeptide as described in any one of claims 8 to 10, or a protein as described in any one of claims 11 to 14.

16. An isolated or purified nucleic acid comprising, from 5' to 3', a first nucleic acid sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence encode the following amino sequences, respectively: SEQ ID NO: 8 and 9; 9 and 8; 10 and 11; 11 and 10; 12 and 13; 13 and 12; 14 and 15; 15 and 14; 22 and 23; 23 and 22; 24 and 25; 25 and 24; 26 and 27; 27 and 26; 28 and 29; 29 and 28; 36 and 37; 37 and 36; 38 and 39; 39 and 38; 40 and 41; 41 and 40; 42 and 43; 43 and 42; 50 and 51; 51 and 50; 52 and 53; 53 and 52; 54 and 55; 55 and 54; 56 and 57; 57 and 56; 64 and 65; 65 and 64; 66 and 67; 67 and 66; 68 and 69; 69 and 68; 70 and 71; 71 and 70; 78 and 79; 79 and 78; 8 or 99 and 98. 0 and 81; 81 and 80; 82 and 83; 83 and 82; 84 and 85; 85 and 84; 92 and 93; 93 and 92; 94 and 95; 95 and 94; 17. The isolated or purified nucleic acid of claim 16, further comprising a third nucleotide sequence inserted between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide.

18. The isolated or purified nucleic acid of claim 17, wherein the cleavable linker peptide comprises the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 103).

19. A recombinant expression vector comprising the nucleic acid of any one of claims 15 to 18.

20. The recombinant expression vector as described in claim 19, wherein it is a transposon or lentiviral vector.

21. An isolated or purified TCR, polypeptide, or protein encoded by any one of claims 15 to 18 or by any one of claims 19 or 20.

22. An isolated or purified TCR, polypeptide, or protein produced by expression in cells of any one of the nucleic acids of claims 15 to 18 or the vector of claim 19 or 20.

23. Generates expression of human p53 Y220C or person p53 R175H A method for host cells having an antigen-specific TCR with an amino acid sequence, the method comprising contacting the cells in vitro with the vector, provided that the vector of claim 19 or 20 is permitted to be introduced into the cells.

24. An isolated or purified host cell comprising the nucleic acid of any one of claims 15 to 18 or the recombinant expression vector of claim 19 or 20.

25. The host cell of claim 24, wherein the cell is a human lymphocyte.

26. The host cell of claim 24, wherein the cell is selected from T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, natural killer (NK) cells, macrophages, pluripotent cells, and specialized cells.

27. An isolated or purified cell population comprising the host cell of any one of claims 24 to 26.

28. A method for producing a TCR according to any one of claims 1 to 7, 21 or 22, a polypeptide according to any one of claims 8 to 10, 21 or 22, or a protein according to any one of claims 11 to 14, 21 or 22, the method comprising culturing a host cell according to any one of claims 24 to 26 or a host cell population according to claim 27, such that the TCR, polypeptide or protein is produced.

29. A pharmaceutical composition comprising (a) a TCR according to any one of claims 1 to 7, 21 or 22, a polypeptide according to any one of claims 8 to 10, 21 or 22, a protein according to any one of claims 11 to 14, 21 or 22, a nucleic acid according to any one of claims 15 to 18, a recombinant expression vector according to claim 19 or 20, a host cell according to any one of claims 24 to 26 or a host cell population according to claim 27, and (b) a pharmaceutically acceptable vector.

30. A method for detecting the presence of cancer in mammals, the method comprising: (a) A sample containing cells of the said cancer is contacted with a TCR according to any one of claims 1 to 7, 21 or 22, a polypeptide according to any one of claims 8 to 10, 21 or 22, a protein according to any one of claims 11 to 14, 21 or 22, a nucleic acid according to any one of claims 15 to 18, a recombinant expression vector according to claim 19 or 20, a host cell according to any one of claims 24 to 26, a host cell population according to claim 27, or a pharmaceutical composition according to claim 29, thereby forming a complex; and (b) Detect the complex. The detection of the complex indicates the presence of cancer in the mammal.

31. The TCR of any one of claims 1 to 7, 21 or 22, the polypeptide of any one of claims 8 to 10, 21 or 22, the protein of any one of claims 11 to 14, 21 or 22, the nucleic acid of any one of claims 15 to 18, the recombinant expression vector of claim 19 or 20, the host cell of any one of claims 24 to 26, the host cell population of claim 27 or the pharmaceutical composition of claim 29 for use in inducing an immune response against cancer in mammalian cells.

32. The TCR of any one of claims 1 to 7, 21 or 22, the polypeptide of any one of claims 8 to 10, 21 or 22, the protein of any one of claims 11 to 14, 21 or 22, the nucleic acid of any one of claims 15 to 18, the recombinant expression vector of claim 19 or 20, the host cell of any one of claims 24 to 26, the host cell population of claim 27 or the pharmaceutical composition of claim 29 for use in the treatment or prevention of cancer in mammals.

33. The host cell population for the purpose as described in claim 31 or 32, wherein the cell population is from the mammal itself.

34. The host cell population for the purpose as described in claim 31 or 32, wherein the cell population is an allogeneic mammal.

35. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, host cell population, or pharmaceutical composition for the said use as described in any one of claims 30 to 34, wherein the cancer is epithelial carcinoma.

36. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, host cell population, or pharmaceutical composition for the said use as described in any one of claims 30 to 34, wherein the cancer is cholangiocarcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer (NSCLC), glioblastoma, cervical cancer, head and neck cancer, breast cancer, pancreatic cancer, or bladder cancer.

37. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, host cell population, or pharmaceutical composition for the said use as described in any one of claims 30 to 36, wherein the cancer is known to comprise a Y220C or R175H mutation in human p53.

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