TP53 mutant epitope peptide, specific T cell receptor and application of TP53 mutant epitope peptide and specific T cell receptor

Abstract

Relates to the field of immunology and tumor treatment. Specifically, the invention relates to a TP53R282W mutant epitope peptide and a pMHC complex, an antigen presenting cell expressing the epitope peptide or the pMHC complex, a tumor vaccine containing the epitope peptide or the pMHC complex, and an application of the antigen presenting cell in prevention or treatment of tumors with TP53R282W mutation. The invention also relates to a T cell receptor (TCR) capable of specifically recognizing the TP53R282W mutant, a conjugate and a fusion protein containing the TCR, an immune cell expressing the TCR, a T cell medicine containing the TCR, and application of the T cell receptor and the fusion protein to prevention or treatment of tumors with the TP53R282W mutant.

Description

TP53 mutant epitope peptides and specific t cell receptors and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to International Patent Application PCT / CN2024 / 113329, filed on August 20, 2024, the entire contents of which are incorporated herein by reference.

[0003] SEQUENCE LISTING

[0004] The present application contains a sequence listing and the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0005] The present application relates to the field of immunology and tumor therapy. In particular, the present application relates to TP53R282W mutant epitope peptides and pMHC complexes formed by the epitope peptides complexed with MHC, antigen presenting cells expressing the epitope peptides or pMHC complexes, tumor vaccines comprising them, and their uses for preventing or treating tumors with TP53R282W mutation. The present application also relates to T cell receptors (TCRs) specifically recognizing TP53R282W mutant, conjugates and fusion proteins comprising the TCRs, immune cells expressing the TCRs, and T cell drugs comprising them, and their uses for preventing or treating tumors with TP53R282W mutation. BACKGROUND

[0006] The TP53 (Tumor protein p53) gene was first discovered in 1979 and is involved in genomic DNA damage repair, is one of the transcription factors regulating apoptosis, and is an important tumor suppressor gene. The p53 protein is composed of five domains, two N-terminal transactivation domains (TAD I, 1-42 and TAD II, 43-62); proline domain (PRD, 64-92); central sequence-specific DNA binding domain (DBD, 102-292); tetramerization domain (OD, 323-356) and C-terminal regulatory domain (363-393). In normal cells, DNA damage induces p53 expression to form a tetramer that binds to specific target sequences on the genomic DNA, initiates the transcription and expression of target genes (e.g., p21), inhibits cell division or affects cell metabolic function, and regulates apoptosis or senescence.

[0007] About 50% of tumors in human tumors have TP53 mutations. The tumor types with high mutation frequency are: 90% of uterine cancer, 85% of ovarian cancer, 70% of head and neck cancer, 90% of small cell lung cancer, 65% of pancreatic cancer, 69% of lung adenocarcinoma, 50% of non-small cell lung cancer, etc. TP53 gene mutation makes p53 protein unable to form a tetramer or lose its DNA binding ability, unable to repair DNA damage, lose the biological function of tumor suppressor genes, leading to tumor occurrence. The position of TP53 gene mutation with high frequency is mostly located in its DNA binding domain, and 95% of mutation sites are located in hot spots such as R175, R248, R249, R273, and R282. Tumors containing TP53 mutations often have high invasiveness, and patients have poor surgical prognosis and short survival. So far, drug research targeting TP53 gene mutations has failed, such as TP53 activator APR-246 (Eprenetapopt) and TP53-MDM2 inhibitors. TP53 is a protein expressed in cells, and the research of drugs targeting TP53 has always been dominated by small molecule drug research, and so far no breakthrough has been made. The main reason is that traditional small molecule drugs mostly inhibit the activity of the target to exert their efficacy, but p53 gene mutation makes it lose its biological function, so the early research of small molecule drugs targeting p53 aims to re-activate p53 to exert its biological function. However, with the failure of the phase III clinical trial of TP53 activator APR-246, the research of p53 activators has temporarily made no breakthrough. In addition, the inhibitor RG7388 targeting the intracellular degradation protein MDM2 of TP53 has also been discontinued.

[0008] T cell receptor (TCR) is a specific receptor expressed on the surface of T lymphocytes, which is generated by rearrangement of embryonic V, D, and J genes, and exerts its biological function by recognizing the pMHC complex formed by the Major Histocompatibility Complex (MHC) and the epitope peptide. In 2019, Steven Rosenberg et al. found that TP53 gene mutation R175H can be presented on the surface of tumor cells by HLA-A*0201, and isolated human T cell receptors that recognize HLA-A*0201 presenting TP53 R175H mutation (Steven A. Rosenberg, Cancer Immunol Res, 2019).

[0009] TP53R282W mutation is common in various solid tumors, with a mutation frequency of about 1-4%, such as esophageal cancer, pancreatic cancer, colorectal cancer, lung cancer, cholangiocarcinoma, head and neck cancer, endometrial cancer, etc. The TP53R282W mutation frequency is about 1% in all solid tumors. According to the statistics of WHO, there were 4.77 million new cases of solid tumors in China in 2022, so about 50,000 patients carried TP53R282W gene mutation every year. At present, there is no drug targeting TP53 mutation approved worldwide, so it is urgent to develop new drugs targeting TP53 mutation to provide new treatment options for patients. SUMMARY

[0010] The present application provides epitope peptides of TP53R282W mutant, complexes of the epitope peptides with MHC-I (pMHC), and T cell receptors (TCRs) that specifically recognize the epitope peptides / pMHC complexes, cells and pharmaceutical compositions comprising these epitope peptides, pMHCs, or TCRs, nucleic acids encoding these epitope peptides, pMHCs, or TCRs, vectors and host cells for making these epitope peptides, pMHCs, or TCRs, and methods for using these epitope peptides, pMHCs, or TCRs to treat subjects. The epitope peptides, pMHCs, and TCRs provided by the present application can be used to induce an immune response against tumors containing TP53R282W mutation and thus treat the above-mentioned tumors in subjects. In addition, the epitope peptides and TCRs provided by the present application are MHC-I restricted, which is the dominant allele in the Asian population, and thus are particularly suitable for patients in the Asian region.

[0011] In one aspect, the present application provides an isolated epitope peptide consisting of 10-25 contiguous amino acid residues of a TP53 protein mutant having a R282W substitution, and comprising amino acid residues 273-282 of the TP53 protein mutant.

[0012] In some embodiments, the epitope peptide is capable of being presented by an MHC class-I molecule and associates with the MHC class-I molecule to form an MHC-peptide (pMHC) complex, which is capable of being recognized by a T cell, e.g., by an antigen-specific T cell receptor on a T cell. Preferably, the MHC class-I molecule in the pMHC complex is HLA-B. Preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801.

[0013] In some embodiments, the amino acid residues 273-282 of the TP53R282W mutant have a sequence as set forth in SEQ ID NO: 36.

[0014] In some specific embodiments, the epitope peptide comprises amino acid residues 273-283 or amino acid residues 273-282. Preferably, the amino acid residues 273-283 or amino acid residues 273-282 of the TP53 protein mutant have the sequence as set forth in SEQ ID NO: 22 or 36, respectively.

[0015] In some embodiments, the TP53 protein mutant has the sequence as set forth in SEQ ID NO: 1.

[0016] In one aspect, the present application provides a peptide-MHC (pMHC) complex comprising an epitope peptide disclosed herein and a MHC molecule;

[0017] Preferably, the MHC molecule is a MHC class-I molecule;

[0018] Preferably, the MHC class-I molecule is HLA-B;

[0019] Preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801.

[0020] In some embodiments, the epitope peptide in the pMHC complex is covalently linked to the MHC molecule or associated with the MHC molecule through non-covalent interaction.

[0021] In some embodiments, the pMHC complex is in soluble form or presented on the cell surface.

[0022] In some embodiments, the pMHC complex is linked to other moieties selected from therapeutic moieties, fluorescent or luminescent labels, radioactive labels, nucleic acid probes and contrast agents, antibodies, or enzymes that generate detectable products.

[0023] In one aspect, the present application provides an isolated T cell receptor (TCR) or antigen binding fragment thereof capable of specifically recognizing any one of the epitope peptides disclosed herein or any one of the pMHC complexes disclosed herein. Preferably, the epitope peptide is presented by a MHC class-I molecule; preferably, the MHC class-I molecule is HLA-B; preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801; preferably, the TCR is soluble or membrane-bound; preferably, the TCR is a full-length TCR, a soluble TCR or a single-chain TCR.

[0024] In one aspect, the present application provides an isolated T cell receptor (TCR) or antigen binding fragment thereof capable of specifically recognizing a TP53 protein having a R282W substitution, the TCR or antigen binding fragment thereof comprising an alpha chain variable region (Va) and / or a beta chain variable region (Vp), wherein:

[0025] (a) the Va comprises a CDR1a, a CDR2a, and a CDR3a; and / or

[0026] (b) the Vp comprises a CDR1p, a CDR2p, and a CDR3p;

[0027] Preferably, the TCR is soluble or membrane-bound. The TCR can be a full-length TCR, a soluble TCR, or a single-chain TCR.

[0028] In some embodiments, in the TCR or antigen-binding fragment thereof:

[0029] (i) the CDR1a comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 14;

[0030] (ii) the CDR2a comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 15;

[0031] (iii) the CDR3a comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 16.

[0032] In some embodiments, in the TCR or antigen-binding fragment thereof:

[0033] (i) the CDR1p comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 18;

[0034] (ii) the CDR2p comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 19;

[0035] (iii) the CDR3p comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 20.

[0036] In some embodiments, in the TCR or antigen-binding fragment thereof:

[0037] (a) the Va comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 10; and / or

[0038] (b) the Vp comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 12.

[0039] In some embodiments, the TCR or antigen-binding fragment thereof is capable of specifically recognizing any one of the epitope peptides disclosed herein or any one of the pMHC complexes disclosed herein. Preferably, the epitope peptide is presented by an MHC-Class I molecule. Preferably, the MHC-Class I molecule is HLA-B. Preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801. Preferably, when the TCR or antigen-binding fragment thereof is expressed on the surface of a T cell, the T cell is activated upon co-culturing with a second cell (e.g. an antigen presenting cell) displaying the epitope peptide according to any one of the epitope peptides disclosed herein.

[0040] In one aspect, the present application provides a conjugate comprising a TCR or antigen-binding fragment thereof disclosed herein and an effector moiety conjugated thereto. Preferably, the TCR or antigen-binding fragment thereof is soluble.

[0041] In one aspect, the present application provides a fusion protein comprising a TCR or antigen-binding fragment thereof disclosed herein and an additional peptide or protein. Preferably, the additional peptide or protein comprises an anti-CD3 single-chain antibody, IL-2 and its mutants, human immunoglobulin constant region, microtubule inhibitor conjugate, etc. Preferably, the TCR or antigen-binding fragment thereof is soluble.

[0042] In one aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any one of the epitope peptides disclosed herein, or one or more nucleotide sequences encoding an epitope peptide and a corresponding MHC molecule disclosed herein, or comprising a nucleotide sequence encoding any one of the TCRs or antigen-binding fragments thereof or the variable region of the alpha chain and / or the variable region of the beta chain disclosed herein, or comprising a nucleotide sequence encoding a fusion protein disclosed herein.

[0043] In one aspect, the present application provides a vector comprising a nucleic acid molecule according to the present application. Preferably, the vector is a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a baculoviral vector.

[0044] In one aspect, the present application provides a host cell comprising a nucleic acid molecule or a vector disclosed herein. Preferably, the host cell comprises a nucleotide sequence encoding an epitope peptide according to the disclosure herein. Preferably, the host cell comprises a nucleotide sequence encoding a TCR or an antigen-binding fragment thereof or an alpha chain variable region and / or a beta chain variable region thereof according to the disclosure herein. Preferably, the host cell comprises a nucleotide sequence encoding a fusion protein according to the disclosure herein. Preferably, the host cell comprises an E. coli, a yeast, an insect cell, or a mammalian cell.

[0045] In one aspect, the present application provides a method of making an epitope peptide, a TCR or an antigen-binding fragment thereof, or a fusion protein disclosed herein, comprising:

[0046] culturing a host cell disclosed herein under conditions that allow expression of the protein, and

[0047] recovering the epitope peptide, or the TCR or the antigen-binding fragment thereof, or the fusion protein from the cultured host cell culture.

[0048] In one aspect, the present application provides an engineered antigen presenting cell (APC) presenting on the cell surface any one of the epitope peptides disclosed herein or any one of the pMHC complexes disclosed herein. Preferably, the epitope peptide is presented by an MHC-Class I molecule. Preferably, the MHC-Class I molecule is HLA-B. Preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801. Preferably, the APC is selected from a dendritic cell, a monocyte, a macrophage, a lymphoblast cell line, or any combination thereof. Preferably, the APC is HLA-B*5701 positive or HLA-B*5801 positive. Preferably, the APC is isolated from an HLA-B*5701 positive subject or an HLA-B*5801 positive subject.

[0049] In one aspect, the present application provides a method of making an engineered APC disclosed herein, comprising:

[0050] (a) providing an APC from a subject; and

[0051] (b) contacting the APC in vitro with any one of the epitope peptides disclosed herein or any one of the pMHC complexes disclosed herein or introducing into the APC an expression vector comprising a nucleotide sequence encoding the epitope peptide disclosed herein or the pMHC complex disclosed herein, to obtain an APC presenting on the surface thereof the epitope peptide or the pMHC complex.

[0052] In one aspect, the present application provides an engineered immune cell expressing on the cell surface any one of the TCRs or antigen-binding fragments thereof disclosed herein. Preferably, the engineered immune cell comprises a nucleotide sequence encoding any one of the TCRs or antigen-binding fragments thereof disclosed herein. The immune cell can be a lymphocyte, for example selected from a T cell (e.g., an alpha beta T cell, a gamma delta T cell, or an iPSC-induced T cell), a tumor infiltrating lymphocyte (TIL), a natural killer (NK) cell, a natural killer T (NKT) cell, or any combination thereof.

[0053] In one aspect, the present application provides a method of making an engineered immune cell according to the present application, comprising:

[0054] (a) providing an immune cell from a subject; and

[0055] (b) introducing into the immune cell of step (a) an isolated nucleic acid molecule or a vector disclosed herein, said nucleic acid molecule or vector comprising a nucleotide sequence encoding a TCR or antigen-binding fragment thereof disclosed herein, to obtain an immune cell expressing said TCR or antigen-binding fragment thereof.

[0056] Preferably, in step (a), the immune cell is pre-treated; the pre-treatment comprises sorting, activating and / or proliferating the immune cell. The pre-treatment can comprise contacting the immune cell with an anti-CD3 antibody and / or an anti-CD28 antibody, thereby stimulating the immune cell and inducing its proliferation, thereby generating a pre-treated immune cell.

[0057] In one aspect, the present application provides a pharmaceutical composition comprising any one of the epitope peptides disclosed herein, any one of the pMHC complexes disclosed herein, a nucleic acid molecule or a vector comprising a nucleotide sequence encoding said epitope peptide or pMHC complex, or a host cell, or an engineered antigen presenting cell (APC) disclosed herein; and a pharmaceutically acceptable carrier and / or excipient. Preferably, the pharmaceutical composition is a tumor vaccine. Preferably, the pharmaceutical composition comprises an adjuvant. Preferably, the pharmaceutical composition further comprises an additional therapeutic agent, for example an anti-tumor agent or an immune-enhancing agent.

[0058] In some embodiments, the anti-tumor agent is selected from an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an anti-metabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, or a TIM3 antibody).

[0059] In some embodiments, the immune potentiator is selected from an immunostimulatory antibody (e.g., an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD40L (CD154) antibody, an anti-41BB (CD137) antibody, an anti-OX40 antibody, an anti-GITR antibody, or any combination thereof) or an immunostimulatory cytokine (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-gamma, IL-10, TGF-beta, GM-CSF, or any combination thereof).

[0060] In one aspect, the present application provides a pharmaceutical composition comprising a TCR or antigen-binding fragment thereof, a conjugate, a fusion protein, a nucleic acid molecule or a vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein, or an engineered immune cell as disclosed herein; and a pharmaceutically acceptable carrier and / or excipient;

[0061] Preferably, the pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immune potentiator.

[0062] In some embodiments, the anti-tumor agent is selected from an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, or a TIM3 antibody).

[0063] In some embodiments, the immune potentiator is selected from an immunostimulatory antibody (e.g., an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD40L (CD154) antibody, an anti-41BB (CD137) antibody, an anti-OX40 antibody, an anti-GITR antibody, or any combination thereof) or an immunostimulatory cytokine (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-gamma, IL-10, TGF-beta, GM-CSF, or any combination thereof).

[0064] In one aspect, the present application provides a method of inducing an immune response against a tumor having a TP53R282W mutation in a subject, and / or preventing or treating a tumor having a TP53R282W mutation in a subject, comprising administering to the subject an epitope peptide disclosed herein, a pMHC complex disclosed herein, a nucleic acid molecule or a vector or a host cell comprising a nucleotide sequence encoding the epitope peptide or pMHC complex, or an engineered antigen presenting cell (APC), or a pharmaceutical composition. In one aspect, the present application provides an epitope peptide disclosed herein, a pMHC complex disclosed herein, a nucleic acid molecule or a vector or a host cell comprising a nucleotide sequence encoding the epitope peptide or pMHC complex, or an engineered antigen presenting cell (APC), or a pharmaceutical composition for use as a medicament. In one aspect, the present application provides the use of an epitope peptide disclosed herein, a pMHC complex disclosed herein, a nucleic acid molecule or a vector or a host cell comprising a nucleotide sequence encoding the epitope peptide or pMHC complex, or an engineered antigen presenting cell (APC), or a pharmaceutical composition for the manufacture of a medicament for inducing an immune response against a tumor having a TP53R282W mutation in a subject, and / or preventing or treating a tumor having a TP53R282W mutation in a subject. The tumor can be selected from, but not limited to, uterine cancer, ovarian cancer, head and neck cancer, small cell lung cancer, pancreatic cancer, lung adenocarcinoma and non-small cell lung cancer. Preferably, the subject is a human. Preferably, the subject is HLA-B*5701 positive or HLA-B*5801 positive. Preferably, the epitope peptide, nucleic acid molecule or vector or host cell, engineered antigen presenting cell (APC), or pharmaceutical composition is administered in combination with, e.g. simultaneously, separately or sequentially, another therapeutic agent; preferably the other therapeutic agent is an immunostimulatory agent or an anti-tumor agent.

[0065] In one aspect, the present application provides a method of inducing an immune response against a tumor having a TP53R282W mutation in a subject, and / or preventing or treating a tumor having a TP53R282W mutation in a subject, comprising administering to the subject a TCR or antigen-binding fragment thereof, a conjugate, a fusion protein, a nucleic acid molecule or a vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein, or an engineered immune cell, or a pharmaceutical composition as disclosed herein. In one aspect, the present application provides a TCR or antigen-binding fragment thereof, a conjugate, a fusion protein, a nucleic acid molecule or a vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein, or an engineered immune cell, or a pharmaceutical composition as disclosed herein for use as a medicament. In one aspect, the present application provides the use of a TCR or antigen-binding fragment thereof, a conjugate, a fusion protein, a nucleic acid molecule or a vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein, or an engineered immune cell, or a pharmaceutical composition as disclosed herein for the manufacture of a medicament for inducing an immune response against a tumor having a TP53R282W mutation in a subject, and / or preventing or treating a tumor having a TP53R282W mutation in a subject; wherein the nucleic acid molecule, vector or host cell comprises a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein. The tumor having a TP53R282W mutation can be selected from, but not limited to, uterine cancer, ovarian cancer, head and neck cancer, small cell lung cancer, pancreatic cancer, lung adenocarcinoma and non-small cell lung cancer. Preferably, the subject is a human. Preferably, the subject is HLA-B*5701 positive or HLA-B*5801 positive.

[0066] In some embodiments, the TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule or vector or host cell, engineered immune cell, or pharmaceutical composition is administered in combination with, e.g. simultaneously, separately or sequentially, another therapeutic agent; preferably the other therapeutic agent is an immunostimulatory agent or an anti-tumor agent. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 shows the results of IFNy release from TIL cells co-cultured with APCs that were electroporated with TP53R282W mRNA in Example 3. The abscissa is each TIL cell prepared in Example 1.

[0068] Figure 2 shows the results of IFNy specific release from T cells expressing different TCR clones in Example 4. The abscissa is each TCR clone constructed based on the sequence of R85.15 TCR.

[0069] Figure 3 shows the results of IFNy release after co-culture of each LCL cell line loaded with different peptide segments and R85.15.1 TCR-T cells in Example 5. "pR282" refers to loading with wild-type TP53 peptide.

[0070] Figure 4 shows the results of IFNy release after co-culture of different tissue- derived tumor cell lines co-expressing HLA-B*5801 and TP53-R282W and R85.15.1 TCR-T cells in Example 6.

[0071] Figure 5 shows the results of IFNy release assay of different R282W peptide segments inducing R85.15.1 TCR-T in Example 7.

[0072] Figure 6 shows the results of avidity testing of R85.15.1 TCR-T to TP53 R282W mutants (reflected as IFNy release) in Example 8.

[0073] Figure 7 shows the results of avidity testing of R85.15.1 TCR-CD4 T or R85.15.1 TCR-CD8 T cells to TP53 R282W mutants (reflected as IFNy release) in Example 8.

[0074] DETAILED DESCRIPTION

[0075] In the present application, unless otherwise indicated, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents, patent applications, and other publications cited herein are fully incorporated by reference herein in their entirety. Also, the practice of the present application employs, unless otherwise indicated, conventional immunological, molecular biological, biochemical, nucleic acid chemical, cell and tissue culture, and recombinant DNA techniques, which are within the skill of the art. The definitions herein prevail in case of conflict with the definitions as set forth in the patents, patent applications, and other publications incorporated by reference herein.

[0076] As used herein, the terms "T cell receptor" and "TCR" are used interchangeably and refer to a molecule comprising a CDR or variable region from an alpha beta or gamma delta T cell receptor. Alpha beta TCRs are the predominant TCRs (95%) found on MHC-restricted T cells. The term TCR includes, but is not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking the transmembrane and cytoplasmic regions, single-chain TCRs containing TCR variable regions connected by a flexible linker, TCR chains linked by engineered disulfide bonds, and the like.

[0077] As used herein, the term“full-length TCR” refers to a TCR comprising a dimer of a first polypeptide chain and a second polypeptide chain, each of which includes a TCR variable region and a TCR constant region including a TCR transmembrane region and a TCR cytoplasmic region. In certain embodiments, a full-length TCR includes a mature full-length TCR a chain and a mature full-length TCR b chain. In certain embodiments, a full-length TCR includes a mature full-length TCR g chain and a mature full-length TCR d chain.

[0078] As used herein, the term“TCR variable region” refers to the portion of a mature TCR polypeptide chain (e.g., a TCR a chain or b chain) that is not encoded by the TRAC gene of a TCR a chain, the TRBC1 gene or TRBC2 gene of a TCR b chain, the TRDC gene of a TCR d chain, or the TRGC1 gene or TRGC2 gene of a TCR g chain. In certain embodiments, a TCR variable region of a TCR a chain encompasses all amino acids of a mature TCR a chain polypeptide encoded by a TRAV gene and / or a TRAJ gene, and a TCR variable region of a TCR b chain encompasses all amino acids of a mature TCR b chain polypeptide encoded by a TRBV gene, a TRBD gene, and / or a TRBJ gene (see, e.g., T cell receptor Factsbook, (2001), LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated by reference herein in its entirety). A TCR variable region typically includes framework regions (FRs) 1, 2, 3, and 4, and complementarity determining regions (CDRs) 1, 2, and 3. The terms“a chain variable region” and“V a” are used interchangeably herein and refer to the variable region of a TCR a chain. The terms“b chain variable region” and“V b” are used interchangeably and refer to the variable region of a TCR b chain.

[0079] As used herein, the term "CDR" or "complementarity determining region" with respect to a TCR refers to the non-contiguous antigen binding sites found within the variable region of a TCR chain (e.g., alpha or beta chain). These regions have been described in LeFranc, (1999), The Immunologist, 7: 132-136; LeFranc et al., (1999), Nucleic Acids Res, 27: 209-212; LeFranc, (2001), T cell receptor Facts book, Academic Press, ISBN 0-12-441352-8; LeFranc et al., (2003), Dev Comp Immunol, 27(1): 55-77; and in Kabat et al., (1991), Sequences of protein of immunological interest, each of which is incorporated herein by reference in its entirety. CDRs can be delineated according to a variety of numbering systems well known in the art, for example, CDRs can be defined by the IMGT numbering system or the Kabat numbering system.

[0080] As used herein, the term "FR" or "framework region" with respect to a TCR refers to those amino acid residues in the variable region of a TCR chain (e.g., alpha or beta chain) other than the CDRs as defined above.

[0081] As used herein, the term "constant region" with respect to a TCR refers to the portion of a TCR encoded by the TRAC gene (for TCR alpha chain), TRBC1 or TRBC2 gene (for TCR beta chain), TRDC gene (for TCR delta chain), or TRGC1 or TRGC2 gene (for TCR gamma chain), optionally lacking all or a portion of the transmembrane region and / or all or a portion of the cytoplasmic region. In certain embodiments, the TCR constant region lacks the transmembrane region and the cytoplasmic region.

[0082] As used herein, the term "antigen binding portion" with respect to a TCR refers to any portion or fragment of a TCR that retains a biological activity of the TCR (parent TCR) as part of the TCR. The biological activity can include the ability to specifically bind the same antigen (e.g., TP53R282W mutant) or MHC-antigen complex bound by the parent TCR.

[0083] As used herein, the "avidity" of a T cell is a biological measure used to describe how a T cell senses its antigen. The avidity of a T cell can be determined in vitro by quantitatively assessing a given T cell functional response (e.g., cytotoxic activity, IFNy production, or proliferation, etc.) upon exposure to increasing doses of antigenic peptide, and quantified by the concentration of antigenic peptide required to induce a half-maximal response (i.e., EC50).

[0084] As used herein, the term "immune cell" refers to any cell of the immune system having one or more effector functions. Immune cells typically include cells that play a role in the immune response, and they generally have hematopoietic origin. The term "effector function" refers to a specialized function of an immune cell, such as a function or response that enhances or promotes an immune attack on a target cell (e.g., killing of the target cell, or inhibition of its growth or proliferation). For example, an effector function of a T cell can be cytolytic activity or an activity that includes secretion of cytokines. Examples of immune cells include T cells (e.g., alpha / beta T cells and gamma / delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived macrophages, among others.

[0085] The immune cells described herein can be self / syngeneic or non-self (e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "self" refers to a cell from the same subject; "allogeneic" refers to a cell from the same species as the comparison cell but genetically different; "syngeneic" refers to a cell from a different subject that is genetically identical to the comparison cell; and "xenogeneic" refers to a cell from a different species than the comparison cell. In certain embodiments, the immune cells of the present application are autologous or allogeneic.

[0086] As used herein, the term "antigen presenting cell" or "APC" refers to any cell capable of presenting a peptide fragment of a protein associated with a major histocompatibility complex (MHC) molecule on its cell surface. Such cells are well known to those of skill in the art and include, but are not limited to, for example, dendritic cells, monocytes, macrophages, lymphoblastoid cell lines (e.g., B-lymphoblastoid cell line B-LCL), and the like.

[0087] As used herein, the terms "major histocompatibility complex" and "MHC" are used interchangeably and refer to a group of tightly linked genes that determine whether transplanted tissues are identical, are closely associated with immune responses, and include primarily MHC class I molecules and MHC class II molecules. "MHC class I molecules" are dimers composed of an MHC class I alpha heavy chain and a beta2 microglobulin, and T cells expressing CD8 molecules react with MHC class I molecules. "MHC class II molecules" are dimers composed of an MHC class II alpha chain and an MHC class II beta chain, and T cells expressing CD4 molecules react with MHC class II molecules. The MHC in humans is referred to as the human leukocyte antigen (HLA) complex.

[0088] As used herein, the terms "MHC-peptide complex," "MHC-peptide complex," or "pMHC complex" are used interchangeably and refer to an MHC molecule (MHC class I or MHC class II) that includes a peptide in the MHC peptide binding pocket as is recognized in the art. In some cases, the MHC molecule can be a membrane-bound protein expressed on the surface of a cell. In other cases, the MHC molecule can be a soluble protein that lacks a transmembrane region or a cytosolic region.

[0089] In the context of a TCR, the terms "specifically binds" or "specifically recognizes" refer to the ability of a TCR to preferentially bind a particular antigen (e.g., a particular peptide or a particular peptide-MHC complex combination). Typically, a TCR that specifically binds to an antigen does not bind or binds with lower affinity to other antigens. In certain embodiments, a TCR or antigen-binding fragment thereof disclosed herein specifically binds to a TP53R282W mutant. In certain embodiments, a TCR or antigen-binding fragment thereof disclosed herein specifically binds to an epitope peptide described herein. In certain embodiments, a TCR or antigen-binding fragment thereof disclosed herein specifically binds to a sequence set forth in any one of SEQ ID NOs: 7, 22, 36.

[0090] As used herein, the term "co-receptor" with respect to a TCR refers to a protein possessed by the surface of a T cell to assist the TCR in recognizing an antigen and participating in the transduction of a T cell activation signal, including CD4 and CD8, which stabilize the TCR-peptide-MHC complex by binding to MHC class II or class I molecules, respectively, and enhance specific T cell responses by bringing a tyrosine kinase (member of the Src family, Lck) near the cytoplasmic tails of the CD3 and zeta proteins, thereby facilitating signal transduction and cell activation. T cells bearing CD4 (CD4 T) are known as T helper cells, and T cells bearing CD8 (CD8 T) have a killing function, such as inducing apoptosis of virus-infected cells, and are also known as cytotoxic T lymphocytes (CTLs).

[0091] As used herein, the term "epitope" refers to a local region of an antigen (e.g., a peptide or a peptide-MHC complex) to which a TCR can bind, also known as an antigenic determinant. The epitope to which a TCR binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA assays, hydrogen / deuterium exchange mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), flow cytometry analysis, mutagenesis mapping (e.g., site-directed mutagenesis mapping), and / or structural modeling. In certain embodiments, the antigen is a peptide-MHC complex or a peptide presented by an MHC molecule.

[0092] As used herein, the term "isolated" refers to having been separated or purified from components that naturally accompany it (e.g., nucleic acids, proteins, or other naturally occurring biological or organic molecules).

[0093] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in both sequences being compared is occupied by the same base or nucleotide monomer subunit (e.g., a certain position in each of two DNA molecules is occupied by adenine, or a certain position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions in the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched, then the two sequences have 60% identity. Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such alignments can be achieved by using methods well known to those skilled in the art, e.g., by the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently accomplished by computer program, e.g., the Align program (DNAstar, Inc.).

[0094] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. A vector that is capable of mediating the expression of an inserted polynucleotide is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the host cell takes up, and expresses, the genetic material carried by the vector. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; bacteriophages; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 -derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0095] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or B. subtilis, eukaryotic cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells, immune cells (such as T lymphocytes, NK cells, monocytes, macrophages or dendritic cells). A host cell can include a single cell or a population of cells.

[0096] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is compatible, philosophically and / or physiologically, with the subject and the active ingredient, is well known in the art, and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerin), and the like. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired properties of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactose, albumin, or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., a phosphate buffered solution), Ringer's solution, and any combination thereof.

[0097] As used herein, the term "prevention" refers to a method carried out in order to stop or delay the occurrence of a disease or disorder or a symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method carried out in order to obtain a beneficial or desired clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0098] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount that is sufficient to prevent, hinder, or delay the occurrence of the disease (e.g., a tumor); an effective amount for treating a disease refers to an amount that is sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight, and gender, the mode of administration of a drug, and other therapies being administered concurrently, etc.

[0099] As used herein, the term "subject" refers to a mammal, such as a primate, e.g., a human. In certain embodiments, the term "subject" refers to a living organism in which an immune response can be elicited. In certain embodiments, the subject (e.g., a human) has a TP53R282W mutation-positive tumor, or is at risk of having the above-mentioned disease.

[0100] Epitope peptides and pMHC complexes

[0101] The present application developed a TCR capable of specifically recognizing a specific mutation R282W in the p53 protein encoded by the TP53 gene, and identified the epitope peptide specifically recognized by the TCR. The sequence of the p53 protein encoded by the TP53 gene is well known to those skilled in the art and can be found in various public databases, one sequence of human wild-type p53 protein is shown in UniProt: P04637-1. The TP53R282W mutant described herein refers to a TP53 mutant in which the Arg amino acid residue at position 282 is mutated to Trp. In this context, when referring to the amino acid positions in the TP53R282W mutant, reference is made to the sequence shown in SEQ ID NO: 1. For example, the expression "amino acid residues 273-282 of the TP53R282W mutant protein" refers to the amino acid residues 273-282 of the sequence shown in SEQ ID NO: 1.

[0102] In some aspects, the present application provides an isolated epitope peptide consisting of 10-25 (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10) consecutive amino acid residues of a TP53R273H mutant and comprising the amino acid residues at positions 273-282 of the TP53R273H mutant. Preferably, the amino acid residues at positions 273-282 of the TP53R273H mutant have the sequence as set forth in SEQ ID NO: 36. In certain embodiments, the TP53R273H mutant has the sequence set forth in SEQ ID NO: 1 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. The epitope peptide can have the ability to be presented by an MHC-Class I molecule and subsequently recognized by a T cell, e.g., by an antigen-specific T cell receptor on a T cell. In some embodiments, the epitope peptide consists of a fragment of SEQ ID NO: 7, in particular any fragment comprising SEQ ID NO: 36, e.g., consists of the sequence set forth in SEQ ID NO: 22.

[0103] In certain embodiments, the epitope peptide of the present application is an MHC-I restricted antigen, i.e., the epitope peptide of the present application can be displayed or presented or form a complex with an MHC-I molecule expressed on the surface of a cell. In certain embodiments, the epitope peptide of the present application is capable of being presented by an MHC-Class I molecule and the epitope peptide associated with an MHC-Class I molecule is capable of being recognized by a T cell, e.g., by an antigen-specific T cell receptor on a T cell. In certain embodiments, the MHC-Class I molecule is HLA-B. In certain embodiments, the HLA-B comprises HLA-B*5701 or HLA-B*5801. Preferably, the HLA-B is HLA-B*5801.

[0104] Thus, in another aspect, the present application provides a MHC-peptide complex (pMHC complex) formed by the epitope peptide of the present application in combination, association, linkage or attachment with a MHC class I molecule. The MHC class I molecule comprises a peptide groove (also known as a peptide binding region) consisting of the a1 and a2 domains of the a chain, capable of binding an epitope peptide with a certain specificity, thereby ensuring that the MHC molecule effectively presents the antigen. In certain embodiments, the MHC class I molecule is HLA-B. There are thousands of alleles of the HLA-B gene encoding HLA-B series antigens. In certain embodiments, the HLA-B comprises HLA-B*5701 or HLA-B*5801, preferably the HLA-B is HLA-B*5801.

[0105] In certain embodiments, the pMHC complex is present on the surface of a cell. Thus, the present application also encompasses a cell expressing the pMHC complex, such as an antigen presenting cell. In certain embodiments, the pMHC complex can be in isolated or substantially purified form. For example, the pMHC complex can be provided in a form that is substantially free of other peptides or proteins. In the context of the present application, the term "MHC molecule" also includes recombinant MHC molecules, non-naturally occurring MHC molecules, and functionally equivalent fragments of MHC, including derivatives or variants thereof, which are capable of providing the retained epitope peptide binding capacity. For example, the MHC molecule can be fused to a therapeutic moiety, attached to a solid support, in soluble form, attached to a tag, biotinylated and / or in multimeric form. The epitope peptide of the present application can be covalently attached or associated by non-covalent interaction to the MHC.

[0106] The epitope peptide and / or pMHC complex of the present application can be linked (covalently or otherwise) to a moiety capable of inducing a therapeutic effect. Such a moiety can be a carrier protein known to be immunogenic, such as keyhole limpet hemocyanin (KLH) and bovine serum albumin, for example, in a vaccine composition. Alternatively, the epitope peptide and / or pMHC complex of the present application can be linked to a fusion partner. The fusion partner can be for detection purposes, or for attaching the epitope peptide or MHC to a solid support, or for MHC oligomerization. The pMHC complex can incorporate a biotinylation site, which can add biotin, for example, using the BirA enzyme (O'Callaghan et al., 1999 Jan 1; 266(1): 9-15). Other suitable fusion partners include, but are not limited to, fluorescent or luminescent labels, radioactive labels, nucleic acid probes and contrast agents, antibodies, or enzymes that produce a detectable product. Detection methods can include flow cytometry, microscopy, electrophoresis or scintillation counting. The fusion partner can include a cytokine, such as interleukin-2, interferon alpha, and granulocyte macrophage colony stimulating factor.

[0107] The pMHC complex of the application can be provided in soluble form or immobilized by attachment to a suitable solid support. Examples of solid supports include, but are not limited to, beads, membranes, agarose, magnetic beads, plates, tubes, columns. The pMHC complex can be attached to an ELISA plate, magnetic beads, surface plasmon resonance biosensor chip, etc. Methods of attaching pMHC complexes to solid supports are known to those skilled in the art and include, for example, the use of affinity binding pairs, such as biotin and streptavidin, or antibody and antigen. Preferably, the pMHC complex can be labeled with biotin and attached to a surface coated with streptavidin.

[0108] The pMHC complex of the application can be in multimeric form, such as a dimer, tetramer, pentamer, or octamer or more. Examples of methods suitable for producing multimeric pMHC complexes are described in Greten et al., Clin. Diagn. Lab Immunol. 2002 Mar; 9(2):216-20, which is incorporated herein by reference in its entirety. Generally, pMHC complex multimers can be produced using pMHC complexes tagged with a biotin residue and complexed by fluorescently labeled streptavidin. Alternatively, multimeric pMHC complexes can be formed by using immunoglobulins as molecular scaffolds. In this system, the extracellular region of the MHC molecule is fused to the constant region of an immunoglobulin heavy chain separated by a short amino acid linker. Carrier molecules such as dextran (WO02072631) can also be used to produce pMHC multimers. Due to avidity effects, multimeric pMHC complexes can be used to improve detection of binding moieties, such as T cell receptors that bind to the complex.

[0109] T cell receptor

[0110] The present application provides an isolated T cell receptor (TCR) or antigen binding fragment thereof capable of specifically recognizing a TP53R282W mutant antigen of the application or an epitope peptide thereof or a corresponding pMHC complex. In certain embodiments, the antigen and epitope peptide thereof are presented by an MHC class I molecule. In certain embodiments, the MHC class I molecule is HLA-B. In certain embodiments, the HLA-B comprises HLA-B*5701 or HLA-B*5801, preferably the HLA-B is HLA-B*5801.

[0111] Optionally, the TCR or antigen-binding fragment thereof is capable of specifically recognizing a TP53R282W mutant, the TCR or antigen-binding fragment thereof comprising an alpha chain variable region (Va) and / or a beta chain variable region (Vp), wherein (a) the Va comprises a CDR1a, a CDR2a, and a CDR3a, (b) the Vp comprises a CDR1p, a CDR2p, and a CDR3p. The TCR can be used in any TCR structure. Optionally, a signal peptide is present at the N-terminus of the variable region Va and / or Vp.

[0112] In certain embodiments, the TCR can be a full-length TCR comprising a full-length alpha chain and a full-length beta chain. In certain embodiments, the TCR is a soluble TCR lacking one or more transmembrane and / or cytoplasmic regions. In certain embodiments, the soluble TCR is produced by fusing the extracellular domain of the TCR of the application to other protein domains (e.g., a maltose binding protein, a thioredoxin, a human constant k domain, or a leucine zipper), see, e.g., et al., Front Oncol., 2015. 1.12; 4:378, which is incorporated by reference herein in its entirety.

[0113] In certain embodiments, the TCR of the application can also be a single-chain TCR comprising Va and Vp linked by a peptide linker. Such single-chain TCRs can comprise Va and Vp each linked to its TCR constant region. Alternatively, the single-chain TCR can comprise Va and Vp, wherein Va, Vp, or both Va and Vp are not linked to a TCR constant region. Exemplary single-chain TCRs are described in PCT Publication Nos. WO 2003 / 020763, WO 2004 / 033685, and WO 2011 / 044186, each of which is incorporated by reference herein in its entirety.

[0114] In certain embodiments, the TCR of the application can comprise two polypeptide chains (e.g., an alpha chain and a beta chain), wherein the chains have been engineered to each have a cysteine residue that can form an interchain disulfide bond. Thus, the TCR of the application can comprise two polypeptide chains linked by an engineered disulfide bond. Exemplary TCRs with engineered disulfide bonds are described in U.S. Patent Nos. 8,361,794 and 8,906,383, each of which is incorporated by reference herein in its entirety.

[0115] In certain embodiments, the T cell receptor disclosed herein is a membrane-bound or soluble T cell receptor. In certain embodiments, the T cell receptor disclosed herein is a full-length TCR, a soluble TCR, or a single-chain TCR.

[0116] In certain embodiments, a TCR or antigen-binding fragment thereof disclosed herein has one or more of the following:

[0117] (i) a CDR1a comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 14;

[0118] (ii) a CDR2a comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 15;

[0119] (iii) a CDR3a comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 16;

[0120] (iv) a CDR1b comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 18;

[0121] (v) a CDR2b comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 19;

[0122] (vi) a CDR3b comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 20.

[0123] In certain embodiments, the Va comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 10, and the Vb comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 12.

[0124] In certain embodiments, the TCR or antigen-binding fragment thereof of the application comprises a Va having the sequence set forth in SEQ ID NO: 10 and / or a Vp having the sequence set forth in SEQ ID NO: 12. In certain embodiments, the TCR or antigen-binding fragment thereof of the application comprises a CDR1a, CDR2a, and CDR3a in Va as set forth in SEQ ID NO: 10, and / or a CDR1p, CDR2p, and CDR3p in Vp as set forth in SEQ ID NO: 12.

[0125] In certain embodiments, the TCR or antigen-binding fragment thereof is capable of specifically recognizing a TP53R282W mutant antigen of the application or a fragment thereof (also referred to as an epitope peptide), particularly an epitope peptide comprising SEQ ID NO: 36. In certain embodiments, the epitope peptide is presented by an MHC-Class I molecule. In certain embodiments, the MHC-Class I molecule is HLA-B. In certain embodiments, the HLA-DP comprises HLA-B*5701 or HLA-B*5801, preferably the HLA-B is HLA-B*5801.

[0126] In certain embodiments, when the TCR or antigen-binding fragment thereof is expressed on the surface of a T cell, the T cell is activated upon co-culture with a second cell displaying (e.g., under the context of an MHC-I, displaying) an epitope peptide of the application. In certain embodiments, the activation of a T cell can be measured using any suitable indicator known in the art. Non-limiting examples of such suitable indicators include increased cytokine (e.g., IL-2, IFNy, etc.) secretion levels, proliferative activity, and / or activation marker (e.g., CD25, CD69, CD107a, etc.) expression levels. In certain embodiments, the activation of a T cell also includes the T cell inducing apoptosis or death of a second cell displaying (e.g., under the context of an MHC-I, displaying) an epitope peptide of the application.

[0127] Conjugates and fusion proteins

[0128] The present application provides a conjugate comprising a TCR or antigen-binding fragment thereof described herein and an effector moiety conjugated thereto.

[0129] In this context, the term "effector moiety" refers to a component or functional group capable of modulating (e.g., increasing or decreasing) a natural activity of a molecule to which it is attached or conferring a novel activity to the molecule. In some embodiments, the effector moiety is a biologically active compound or polypeptide (e.g., a compound or polypeptide having an effector function for a cell targeted by the TCR), or a detectable label.

[0130] In this context, the term "conjugation" refers to any method known in the art for functionally linking protein domains, including but not limited to: recombinant fusion with or without a linker, intein-mediated fusion, non-covalent binding, and covalent bonding, such as disulfide bonding, peptide bonding, hydrogen bonding, electrostatic bonding, and conformational bonding, such as biotin-avidin binding. In certain embodiments, conjugation with an effector moiety can be performed chemically or recombinantly, the chemical being the formation of a covalent bond between two molecules to form one molecule.

[0131] In certain embodiments, the effector moiety can be a therapeutic moiety. A therapeutic moiety refers to a compound or polypeptide that can be used as a therapeutic agent. The conjugate takes advantage of the targeting nature of the TCR to allow the therapeutic moiety to exert a therapeutic effect on the cell targeted by the TCR.

[0132] In certain embodiments, the TCR or antigen-binding fragment thereof of the present application is optionally conjugated with an effector moiety via a linker, such as a peptide linker. In certain embodiments, the effector moiety is linked to the N- or C-terminus of the TCR or antigen-binding fragment thereof of the present application.

[0133] In certain embodiments, when the effector moiety is a peptide or protein, the conjugate is preferably a fusion protein.

[0134] Accordingly, the present application also provides a fusion protein comprising the TCR or antigen-binding fragment thereof disclosed herein and an additional peptide or protein.

[0135] In certain embodiments, the TCR or antigen-binding fragment thereof of the present application is optionally fused with an additional peptide or protein via a peptide linker. In certain embodiments, the additional peptide or protein is linked to the N- or C-terminus of the TCR or antigen-binding fragment thereof of the present application.

[0136] In certain embodiments, the additional peptide or protein can be selected from the various peptide or protein effector moieties described above.

[0137] Preparation of epitope peptides, pMHC complexes, TCRs, and fusion proteins

[0138] The epitope peptides, pMHC complexes, TCRs, or fusion proteins comprising the TCRs of the present application can be prepared in various methods known in the art, such as by genetic engineering recombinant technology. For example, a DNA molecule encoding them is obtained by chemical synthesis or PCR amplification; the resulting DNA molecule is inserted into an expression vector, which is then transfected into a host cell; then, the transfected host cell is cultured under specific conditions, and the epitope peptides, pMHC complexes, TCRs, or fusion proteins comprising the TCRs of the present application are expressed.

[0139] Accordingly, the present application provides an isolated nucleic acid molecule comprising one or more of:

[0140] (i) a nucleotide sequence encoding an epitope peptide disclosed herein;

[0141] (ii) a nucleotide sequence encoding a TCR or antigen-binding fragment thereof or an alpha chain variable region and / or a beta chain variable region thereof disclosed herein;

[0142] (iii) a nucleotide sequence encoding a fusion protein comprising the TCR or antigen-binding fragment thereof;

[0143] (iv) a nucleotide sequence encoding an epitope peptide disclosed herein and a nucleotide sequence of an MHC molecule capable of forming a pMHC complex with the epitope peptide. In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding an alpha chain variable region of a TCR or antigen-binding fragment thereof disclosed herein and a second nucleotide sequence encoding a beta chain variable region thereof. In certain embodiments, the first and second nucleotide sequences are optionally linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A) or an internal ribosome entry site (IRES). In certain embodiments, the self-cleaving peptide is P2A.

[0144] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the TCR alpha chain variable region and a second nucleotide sequence encoding the TCR beta chain variable region.

[0145] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the alpha chain of the TCR or antigen-binding fragment thereof and a second nucleotide sequence encoding the beta chain of the TCR or antigen-binding fragment thereof.

[0146] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules.

[0147] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present on the same isolated nucleic acid molecule in any order; in certain embodiments, the first nucleotide sequence and the second nucleotide sequence are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A) in any order.

[0148] In certain embodiments, the isolated nucleic acid molecule comprises a third nucleotide sequence encoding an epitope peptide disclosed herein and a fourth nucleotide sequence encoding an MHC molecule capable of forming a pMHC complex with the epitope peptide. Preferably, the MHC molecule is an MHC class I molecule, such as HLA-B*5801 or HLA-B*5701.

[0149] In certain embodiments, the third nucleotide sequence and the fourth nucleotide sequence are present on different isolated nucleic acid molecules.

[0150] In certain embodiments, the third nucleotide sequence and the fourth nucleotide sequence are present on the same isolated nucleic acid molecule in any order; in certain embodiments, the third nucleotide sequence and the fourth nucleotide sequence are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A) in any order.

[0151] In some aspects, the present application provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In certain embodiments, the vector of the present application is, e.g., a plasmid, a cosmid, a phage, etc. In certain embodiments, the vector comprises a nucleotide sequence encoding a TCR or antigen-binding fragment thereof or a variable region of an alpha chain and / or a variable region of a beta chain thereof or an alpha chain and / or a beta chain thereof as described above.

[0152] In certain embodiments, the vector comprises a first nucleotide sequence encoding a TCR alpha chain variable region and a second nucleotide sequence encoding a TCR beta chain variable region.

[0153] In certain embodiments, the vector comprises a first nucleotide sequence encoding an alpha chain of a TCR or antigen-binding fragment thereof and a second nucleotide sequence encoding a beta chain of the TCR or antigen-binding fragment thereof.

[0154] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present on different vectors. In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present on the same vector in any order. In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A) in any order.

[0155] In certain embodiments, the vector comprises a nucleotide sequence encoding a fusion protein as described above.

[0156] In certain embodiments, the vector comprises a third nucleotide sequence encoding an epitope peptide disclosed herein and a fourth nucleotide sequence encoding an MHC molecule capable of forming a pMHC complex with the epitope peptide. Preferably, the MHC molecule is an MHC class-I molecule. In certain embodiments, the third nucleotide sequence and the fourth nucleotide sequence are present on different vectors. In certain embodiments, the third nucleotide sequence and the fourth nucleotide sequence are present on the same vector in any order; in certain embodiments, the third nucleotide sequence and the fourth nucleotide sequence are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A) in any order.

[0157] In certain embodiments, the vector is a viral vector, such as a lentivirus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, or a baculovirus vector.

[0158] In some aspects, the present application provides a host cell comprising an isolated nucleic acid molecule disclosed herein or a vector disclosed herein. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, e.g., mouse cells, human cells, etc.).

[0159] In another aspect, there is also provided a method of making an epitope peptide, a pMHC complex, a TCR, or a fusion protein comprising a TCR of the present application, comprising, (1) culturing a host cell as described above under conditions that allow expression of the protein, and (2) recovering the epitope peptide, the pMHC complex, the TCR, or the fusion protein comprising a TCR from the cultured host cell culture.

[0160] The peptide epitope and the MHC molecule comprised in the pMHC complex of the present application can be expressed by the same cell or by separate cells. In certain embodiments, the method of making a pMHC complex of the present application further comprises separately making the epitope peptide and the MHC molecule and recovering, and then allowing the epitope peptide and the MHC molecule to bind or associate under suitable conditions to form the pMHC complex. In certain embodiments, the pMHC complex can be further stabilized, for example, by introducing a non-native linkage between the epitope peptide and the MHC molecule, preferably, the non-native linkage is a covalent linkage, such as a disulfide bond linkage.

[0161] Engineered antigen presenting cells (APCs)

[0162] The epitope peptides of the present application can be used in T cell-based immunotherapy. In some cases, T cell antigens recognize MHC-peptide complexes presented on the surface of APCs through their TCRs to induce MHC-restricted immune responses against TP53 mutants. Accordingly, the present application provides an engineered antigen presenting cell (APC) that presents an epitope peptide disclosed herein on the cell surface.

[0163] In certain embodiments, the epitope peptide is presented by an MHC class I molecule. In certain embodiments, the MHC class I molecule is HLA-B. In certain embodiments, the HLA-B comprises HLA-B*5701 or HLA-B*5801, preferably, the HLA-B is HLA-B*5801. The APC can be selected from a dendritic cell, a monocyte, a macrophage, a lymphoblastoid cell (e.g., a B lymphoblastoid cell, B-LCL), or any combination thereof.

[0164] In certain embodiments, the APC is HLA-B*5701 positive or HLA-B*5801 positive. In certain embodiments, the APC is isolated from an HLA-B*5701 positive subject or an HLA-B*5801 positive subject. The engineered APC antigen is obtained by contacting the APC with an epitope peptide disclosed herein in vitro (i.e., exposing the APC to a sufficient amount of the epitope peptide). Alternatively, the engineered APC is obtained by introducing into the APC an expression vector encoding a nucleotide sequence of an epitope peptide disclosed herein in vitro.

[0165] Optionally, the APC can be self / allogeneic or non-self (e.g., allogeneic). By "self" is meant a cell from the same subject; by "allogeneic" is meant a cell of the same species that is genetically different from the comparison cell. The APC can be isolated or obtained from any tissue where such cells are found, or can be otherwise cultured and provided. For example, the APC can be found in the bone marrow or peripheral blood mononuclear cells (PBMCs) of a mammal, in the spleen of a mammal, or in the skin of a mammal (i.e., Langerhans cells, which are antigen presenting cells in the skin and mucosa, can be found in the skin), and then cultured in a medium containing appropriate cytokines and sorted to obtain the APC.

[0166] Accordingly, the present application also relates to a method of making the engineered APC described above, comprising: (1) providing an APC from a subject; (2) contacting the APC with an epitope peptide disclosed herein in vitro or introducing into the APC an expression vector comprising a nucleotide sequence encoding an epitope peptide disclosed herein to obtain an APC presenting the epitope peptide on its surface.

[0167] Engineered immune cells

[0168] The TCR or antigen-binding fragment thereof of the application can be used in T cell-based immunotherapy. In some cases, T cells expressing the TCR of the application induce an MHC-restricted immune response to TP53 mutants by recognizing MHC-peptide complexes. Accordingly, the present application provides engineered immune cells expressing a TCR or antigen-binding fragment thereof disclosed herein on the cell surface. The engineered immune cells of the application have antigenic specificity for TP53R282W mutants. In certain embodiments, the engineered immune cells of the application have one or more features selected from the following:

[0169] (i) specifically bind to TP53R282W mutants, but not to, or with lower affinity to, other TP53 proteins (including wild-type TP53 proteins or other mutants);

[0170] (ii) specifically bind to an epitope peptide disclosed herein (e.g., a sequence set forth in any one of SEQ ID NOs: 7, 22, 36);

[0171] (iii) upon co-culture with an APC displaying (e.g., in the context of MHC-I, displaying) an epitope peptide of the application, the activation includes, without limitation, increased levels of cytokine (e.g., IL-2, IFNy, etc.) secretion, proliferative activity, and / or expression levels of activation markers (e.g., CD25, CD69, CD 107a, etc.), and increased killing activity of a second cell displaying (e.g., in the context of MHC-I, displaying) an epitope peptide of the application.

[0172] In certain embodiments, the engineered immune cells comprise a nucleotide sequence encoding a TCR or antigen-binding fragment thereof disclosed herein.

[0173] The immune cells can be isolated or obtained from any tissue in which such cells are found. For example, immune cells can be found in the peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, or tumors of a mammal, which are then optionally cultured in media containing appropriate cytokines and sorted to obtain the desired immune cells. Alternatively, the immune cells can be cultured and provided in other ways, for example, obtained by induced differentiation from a precursor cell of an immune cell (e.g., a precursor of a T lymphocyte), which can be, for example, a pluripotent stem cell (e.g., an embryonic stem cell, an induced pluripotent stem cell), a hematopoietic stem cell, or a lymphocyte progenitor cell, which is isolated and / or enriched from, for example, bone marrow, umbilical cord blood, or peripheral blood.

[0174] In certain embodiments, the immune cell is a lymphocyte. In certain embodiments, the immune cell is selected from a T cell, a tumor infiltrating lymphocyte (TIL), a natural killer (NK) cell, a natural killer T (NKT) cell, or any combination thereof. Exemplary immune cells that can be used to express a TCR of the application include PBMCs, TILs, and / or T cells. In certain embodiments, the T cell is selected from an alpha beta T cell, a gamma delta T cell, an iPSC-induced T cell, a CD8+ cytotoxic T cell, a CD4+ cytotoxic T cell, a CD4+ helper T cell (e.g., a Thl or Th2 cell), a CD4 / CD8 double positive T cell, a tumor infiltrating T cell, a thymocyte, a memory T cell, an NKT cell, e.g., a constant NKT cell. In certain embodiments, the immune cell comprises a CD8+ T cell. Those of skill in the art will appreciate that an immune cell can also include a progenitor cell (a precursor cell) of an immune cell, wherein the progenitor cell can be induced to differentiate into an immune cell, either in vivo or in vitro. Thus, in certain embodiments, an immune cell includes a progenitor cell of an immune cell, e.g., contains a hematopoietic stem cell within a population of CD34+ cells derived from umbilical cord blood, bone marrow, or flowing peripheral blood, which differentiates into a mature immune cell after administration to a subject, or which can be induced to differentiate into a mature immune cell in vitro.

[0175] Optionally, the immune cell can be self / autologous or non-self (e.g., allogeneic). By "self" is meant a cell from the same subject; by "allogeneic" is meant a cell of the same species that is genetically different from the comparison cell. In certain embodiments, the immune cell is isolated from an HLA-B*5701 positive subject or an HLA-B*5801 positive subject.

[0176] It is to be understood that the engineered immune cells of the application can be comprised in an isolated population of cells. The population of cells can be a heterogeneous population, e.g., the population of cells contains at least one other cell in addition to the engineered immune cell of the application, which other cell does not have antigenic specificity for the TP53R282W mutant, or e.g., the population of cells comprises more than one type of immune cell, but each of these types of immune cells expresses a TCR of the application and thus has antigenic specificity for the TP53R282W mutant. Further, the population of cells can also be a substantially homogeneous population, e.g., wherein the population consists essentially of, or consists of, T cells having antigenic specificity for the TP53R282W mutant.

[0177] Accordingly, the present application also provides a method for preparing the above-mentioned engineered immune cell, comprising: (1) providing an immune cell from a subject; (2) introducing a nucleic acid molecule or a vector comprising a nucleotide sequence encoding the TCR or the antigen-binding fragment thereof of the present application into the immune cell of step (1) to obtain an immune cell expressing the TCR or the antigen-binding fragment thereof.

[0178] In certain embodiments, in step (1), the immune cell is pre-treated; the pre-treatment comprises sorting, activating and / or proliferating the immune cell. In certain embodiments, the pre-treatment comprises contacting the immune cell with an agent selected from the group consisting of an anti-CD3 antibody and / or an anti-CD28 antibody, thereby stimulating the immune cell and inducing its proliferation, thereby generating a pre-treated immune cell.

[0179] In certain embodiments, in step (2), the nucleic acid molecule or the vector can be introduced into the immune cell by various suitable means, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (e.g. liposome) or viral vector-mediated transfection (e.g. lentivirus infection, retrovirus infection, adenovirus infection), and other physical, chemical or biological means for transferring into a host cell, such as transposon technology, CRISPR-Cas9 technology, etc. In certain embodiments, after step (2), the method further comprises: expanding the immune cell obtained in step (2).

[0180] Epitope peptide-based therapy

[0181] The epitope peptide, the pMHC complex comprising the epitope peptide or the APC presenting the epitope peptide of the present application can be used in immunotherapy to induce an anti-tumor immune response.

[0182] Accordingly, the present application provides a first pharmaceutical composition comprising: an epitope peptide disclosed herein, a nucleic acid molecule or a vector comprising a nucleotide sequence encoding the epitope peptide or a host cell, a pMHC complex comprising the epitope peptide, a nucleic acid molecule or a vector comprising a nucleotide sequence encoding the pMHC complex or a host cell, or an engineered antigen presenting cell (APC) disclosed herein; and a pharmaceutically acceptable carrier and / or excipient.

[0183] In certain embodiments, the first pharmaceutical composition is a tumor vaccine. In certain embodiments, the pharmaceutical composition comprises an adjuvant. Adjuvants are those substances that are capable of non-specifically enhancing the immune response, such as Freund's complete adjuvant, Freund's incomplete adjuvant, ligands for Toll receptors, immunostimulatory antibodies (such as anti-CD3 antibodies, anti-CD28 antibodies, anti-CD40L (CD154) antibodies, anti-41BB (CD137) antibodies, anti-OX40 antibodies, anti-GITR antibodies, or any combination thereof) or immunostimulatory cytokines (such as IL-2, IL-3, IL-12, IL-15, IL-18, IFNy, IL-10, TGF-B, GM-CSF, or any combination thereof IL-2), and the like.

[0184] In certain embodiments, the first pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immune-enhancing agent. In certain embodiments, the anti-tumor agent is selected from alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers (such as gemcitabine, 5-fluorouracil, taxanes, cisplatin, and the like), anti-angiogenic agents, cytokines (such as GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, and the like), antibodies that specifically target tumor cells (such as CD20 antibodies like rituximab, Her2 antibodies like trastuzumab, VEGF antibodies like bevacizumab, EGFR antibodies like cetuximab, and the like), immune checkpoint inhibitors (such as PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, or TIM3 antibodies).

[0185] In certain embodiments, the immune-enhancing agent is selected from immunostimulatory antibodies (such as anti-CD3 antibodies, anti-CD28 antibodies, anti-CD40L (CD154) antibodies, anti-41BB (CD137) antibodies, anti-OX40 antibodies, anti-GITR antibodies, or any combination thereof) or immunostimulatory cytokines (such as IL-2, IL-3, IL-12, IL-15, IL-18, IFNy, IL-10, TGF-B, GM-CSF, or any combination thereof).

[0186] In certain embodiments, in the first pharmaceutical composition, the epitope peptide of the application, the pMHC complex comprising the epitope peptide, the engineered APC, and the additional therapeutic agent can be provided as separate components or as a mixed component.

[0187] In another aspect, the present application provides a method for inducing an immune response against a tumor having a TP53R282W mutation in a subject, and / or preventing or treating a tumor having a TP53R282W mutation in a subject, comprising administering to a subject in need thereof an effective amount of an epitope peptide disclosed herein, a nucleic acid molecule or a vector or a host cell comprising a nucleotide sequence encoding the epitope peptide, a pMHC complex comprising the epitope peptide, a nucleic acid molecule or a vector or a host cell comprising a nucleotide sequence encoding the pMHC complex, or an engineered antigen presenting cell (APC) disclosed herein, or the first pharmaceutical composition.

[0188] In certain embodiments, the tumor having a TP53R282W mutation is selected from uterine cancer, ovarian cancer, head and neck cancer, small cell lung cancer, pancreatic cancer, lung adenocarcinoma, non-small cell lung cancer, colorectal adenocarcinoma, etc.

[0189] In certain embodiments, the subject is a human. In certain embodiments, the subject is HLA-B*5701 positive or HLA-B*5801 positive.

[0190] In certain embodiments, the epitope peptide, the nucleic acid molecule or the vector or the host cell comprising a nucleotide sequence encoding the epitope peptide, the pMHC complex comprising the epitope peptide, the nucleic acid molecule or the vector or the host cell comprising a nucleotide sequence encoding the pMHC complex, or the engineered antigen presenting cell (APC) disclosed herein, or the first pharmaceutical composition of the present application can be administered in combination with an additional therapeutic agent, e.g. an immune-enhancing agent or an anti-tumor agent. Thus, in certain embodiments, the method further comprises administering to the subject an additional therapeutic agent, e.g. an immune-enhancing agent or an anti-tumor agent, e.g. simultaneously, separately or sequentially.

[0191] In certain embodiments, the epitope peptide, the nucleic acid molecule or the vector or the host cell comprising a nucleotide sequence encoding the epitope peptide, the pMHC complex comprising the epitope peptide, the nucleic acid molecule or the vector or the host cell comprising a nucleotide sequence encoding the pMHC complex, or the engineered antigen presenting cell (APC) disclosed herein, or the first pharmaceutical composition of the present application can be administered in combination with an additional therapy, e.g. simultaneously, separately or sequentially. Such additional therapy can be any therapy known to be used for tumors, e.g. surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy or palliative therapy.

[0192] The epitope peptides of the present application, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the epitope peptides, pMHC complexes comprising the epitope peptides, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the pMHC complexes, engineered antigen presenting cells (APCs), or pharmaceutical compositions comprising the same, can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The medicaments of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the epitope peptides of the present application, engineered antigen presenting cells (APCs), or pharmaceutical compositions comprising the same, in the required amount in an appropriate solvent with optional simultaneous incorporation of other desired ingredients including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof, followed by sterilization by filtration. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum or freeze-drying) for ease of storage and use. Such sterile lyophilized powders can be reconstituted into sterile injection solutions just prior to use by the dispersion in an appropriate carrier, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH-buffered solutions (e.g., phosphate-buffered solutions), Ringer's solution, and any combination thereof.

[0193] Thus, in certain exemplary embodiments, the first pharmaceutical composition comprises a sterile, injectable liquid, such as an aqueous or non-aqueous suspension or solution. In certain exemplary embodiments, such sterile, injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH-buffered solutions (e.g., phosphate-buffered solutions), Ringer's solution, and any combination thereof.

[0194] The epitope peptides of the application, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the epitope peptides, pMHC complexes comprising the epitope peptides, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the pMHC complexes, engineered antigen presenting cells (APCs), or pharmaceutical compositions comprising the same, can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrafollicular, intracellular reticulum, inguinal, intravesical, local (e.g., powders, salves or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration / mode of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by those skilled in the art that the route of administration and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the epitope peptides of the application, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the epitope peptides, engineered antigen presenting cells (APCs), or pharmaceutical compositions comprising the same are administered by intravenous injection or bolus.

[0195] The first pharmaceutical composition can include a "therapeutically effective amount" or a "prophylactically effective amount" of an epitope peptide of the application, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the epitope peptides, pMHC complexes comprising the epitope peptides, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the pMHC complexes, or engineered antigen presenting cells (APCs). In this context, a "therapeutically effective amount" is an amount capable of producing an immune response in the treated subject that is capable of reducing or inhibiting the proliferation of tumor cells and / or eliminating tumor cells. A "prophylactically effective amount" refers to an amount capable of producing an immune response in the treated subject against target cells (e.g., tumor cells containing a TP53 mutation) that is capable of preventing the formation of a tumor in the subject, or that is capable of substantially reducing the chances of the subject forming or continuing to form a tumor.

[0196] In another aspect, the application provides the use of an epitope peptide disclosed herein, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the epitope peptides, pMHC complexes comprising the epitope peptides, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the pMHC complexes, or engineered antigen presenting cells (APCs) disclosed herein, or the first pharmaceutical composition, for the manufacture of a medicament for inducing an immune response against a tumor having a TP53R282W mutation in a subject, and / or for preventing or treating a tumor having a TP53R282W mutation in a subject.

[0197] TCR-based therapy

[0198] The TCR or immune cell expressing the TCR of the application can be used in T cell-based immunotherapy to achieve killing of tumors containing the TP53R282W mutation. Accordingly, the present application provides a second pharmaceutical composition comprising: a TCR or antigen-binding fragment thereof disclosed herein, a conjugate comprising the TCR or antigen-binding fragment thereof, or a fusion protein comprising the TCR or antigen-binding fragment thereof, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein, or an engineered immune cell disclosed herein; and a pharmaceutically acceptable carrier and / or excipient.

[0199] In certain embodiments, the second pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immune-enhancing agent. In certain embodiments, the anti-tumor agent is selected from an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an anti-metabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer (e.g., gemcitabine, 5-fluorouracil, a taxane, cisplatin, etc.), an anti-angiogenic agent, a cytokine (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), a specific targeting tumor cell antibody (e.g., a CD20 antibody such as rituximab, a Her2 antibody such as trastuzumab, a VEGF antibody such as bevacizumab, an EGFR antibody such as cetuximab, etc.), an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, or a TIM3 antibody).

[0200] In certain embodiments, the immune-enhancing agent is selected from an immunostimulatory antibody (e.g., an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD40L (CD154) antibody, an anti-41BB (CD137) antibody, an anti-OX40 antibody, an anti-GITR antibody, or any combination thereof) or an immunostimulatory cytokine (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFNy, IL-10, TGF-B, GM-CSF, or any combination thereof).

[0201] In certain embodiments, in the second pharmaceutical composition, the TCR or antigen-binding fragment thereof, conjugate, fusion protein, or engineered immune cell of the application can be provided as a separate component or as a mixed component with the additional therapeutic agent.

[0202] In another aspect, the present application provides a method for inducing an immune response against a tumor having a TP53R282W mutation in a subject, and / or preventing or treating a tumor having a TP53R282W mutation in a subject, comprising administering to a subject in need thereof an effective amount of a TCR or antigen-binding fragment thereof disclosed herein, a conjugate comprising the TCR or antigen-binding fragment thereof, or a fusion protein comprising the TCR or antigen-binding fragment thereof, a nucleic acid molecule or a vector or a host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein, or an engineered immune cell disclosed herein, or the second pharmaceutical composition.

[0203] In certain embodiments, the tumor having a TP53R282W mutation is selected from uterine cancer, ovarian cancer, head and neck cancer, small cell lung cancer, pancreatic cancer, lung adenocarcinoma, non-small cell lung cancer, colorectal cancer, and the like.

[0204] In certain embodiments, the subject is a human. In certain embodiments, the subject is HLA-B*5701 positive or HLA-B*5801 positive.

[0205] In certain embodiments, the method comprises: (1) providing an immune cell required by a subject; (2) introducing a nucleotide sequence encoding a TCR or antigen-binding fragment thereof disclosed herein into the immune cell of step (1) to obtain an immune cell expressing the TCR or antigen-binding fragment thereof on the surface; (3) administering the immune cell obtained in step (2) to the subject.

[0206] In certain embodiments, the method comprises a step of obtaining an immune cell from the subject prior to step (1). The immune cell can be isolated or obtained from any tissue where such cells are found (e.g., peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors), or can be otherwise cultured and provided, e.g., obtained from induced differentiation of a precursor cell of an immune cell (e.g., a precursor of a T lymphocyte).

[0207] In certain embodiments, the immune cell is selected from a lymphocyte. In certain embodiments, the immune cell is selected from a T cell (e.g., an alpha beta T cell, a gamma delta T cell, or an iPSC-induced T cell), a tumor infiltrating lymphocyte (TIL), a natural killer (NK) cell, a natural killer T (NKT) cell, or any combination thereof. In certain embodiments, the immune cell comprises a CD8+ T cell.

[0208] In certain exemplary embodiments, peripheral blood mononuclear cells (PBMCs) and / or TILs are obtained from the subject and directly genetically modified to express the TCR.

[0209] In certain exemplary embodiments, T cells are obtained from the subject and genetically modified to express a TCR. T cells can be obtained from a variety of sources, for example, T cells can be obtained from a unit of blood collected from a subject using a variety of techniques known to those of skill in the art, such as sedimentation, for example, FICOLL™ separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The product of apheresis typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing. The washing step can be accomplished by methods known to those of skill in the art, such as by use of a semi-automated flow-through centrifuge, as will be understood by one of ordinary skill in the art. Following washing, the cells can be resuspended in a variety of biocompatible buffers or other salt solutions with or without buffers. In certain embodiments, unwanted components in the apheresis sample can be removed in the medium in which the cells are directly resuspended. In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes (e.g., by centrifugation through a PERCOLL™ gradient). Particular T cell subpopulations expressing one or more of the following markers: CD3, CD4, CD8, and CD137 can be further isolated by positive or negative selection techniques. In one embodiment, particular T cell subpopulations expressing CD3, CD4, CD8, and CD137 are further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies against surface markers specific for the cells being negatively selected. One exemplary method is cell sorting and / or selection by negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. For example, to enrich for CD8+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies against CD14, CD16a, CD16b, CD19, CD36, CD123, CD235a, CD56, and CD4. Flow cytometry and cell sorting can also be used to isolate the desired cell populations for use in the application.

[0210] In certain embodiments, immune cells, such as T cells, can be activated and expanded (or differentiated, in the case of progenitor cells) in vitro prior to genetic modification.

[0211] In certain embodiments, the TCR or antigen-binding fragment thereof disclosed herein, conjugate comprising the TCR or antigen-binding fragment thereof, or fusion protein comprising the TCR or antigen-binding fragment thereof, nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein, or engineered immune cell disclosed herein, or the second pharmaceutical composition can be administered in combination with an additional therapeutic agent, e.g., an immune-enhancing agent or an anti-tumor agent. Thus, in certain embodiments, the method further comprises administering to the subject an additional therapeutic agent, e.g., an immune-enhancing agent or an anti-tumor agent, e.g., simultaneously, separately, or sequentially.

[0212] In certain embodiments, the TCR or antigen-binding fragment thereof disclosed herein, conjugate comprising the TCR or antigen-binding fragment thereof, or fusion protein comprising the TCR or antigen-binding fragment thereof, nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein, or engineered immune cell disclosed herein, or the second pharmaceutical composition can be administered in combination with an additional therapy, e.g., simultaneously, separately, or sequentially. Such additional therapy can be any therapy known to be useful for tumors, e.g., surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.

[0213] The TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein, engineered immune cell, or pharmaceutical composition comprising the same of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The medicament of the present application should be sterile and stable under the conditions of production and storage. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by incorporating the TCR or antigen-binding fragment thereof, conjugate, fusion protein, engineered immune cell, or pharmaceutical composition comprising the same of the present application in the required dose in an appropriate solvent with optional simultaneous incorporation of other desired ingredients including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof, followed by sterilization by filtration. In addition, a sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum or freeze-drying) for ease of storage and use. Such a sterile lyophilized powder can be reconstituted into a sterile injection solution just prior to use by the dispersion in an appropriate carrier, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH-buffered solution (e.g., phosphate-buffered solution), Ringer's solution, and any combination thereof.

[0214] Thus, in certain exemplary embodiments, the second pharmaceutical composition comprises a sterile injectable liquid, such as an aqueous or non-aqueous suspension or solution. In certain exemplary embodiments, such a sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH-buffered solution (e.g., phosphate-buffered solution), Ringer's solution, and any combination thereof.

[0215] The TCR or antigen-binding fragment thereof, conjugate, fusion protein of the application, nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein, engineered immune cell, or pharmaceutical composition comprising the same, of the present application can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrafollicular, intracisternal, inguinal, intravesical, local (e.g., powders, salves or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by those skilled in the art that the route and / or means of administration will vary depending on the intended purpose. In certain embodiments, the TCR or antigen-binding fragment thereof, conjugate, fusion protein of the application, nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein, engineered immune cell, or pharmaceutical composition comprising the same, of the present application is administered by intravenous injection or bolus.

[0216] The second pharmaceutical composition can include a "therapeutically effective amount" or a "prophylactically effective amount" of the TCR or antigen-binding fragment thereof, conjugate, fusion protein of the application, nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein, engineered immune cell, or pharmaceutical composition comprising the same. In this context, a "therapeutically effective amount" is an amount that is capable of producing an immune response in the treated subject that is capable of reducing or inhibiting the proliferation of tumor cells and / or eliminating tumor cells. A "prophylactically effective amount" refers to an amount that is capable of producing an immune response in the treated subject against target cells (e.g., tumor cells containing a TP53 mutation) that is capable of preventing the formation of a tumor in the subject, or that is capable of substantially reducing the chances of the subject forming or continuing to form a tumor.

[0217] In another aspect, the present application provides the use of a TCR or antigen-binding fragment thereof disclosed herein, a conjugate comprising the TCR or antigen-binding fragment thereof, or a fusion protein comprising the TCR or antigen-binding fragment thereof, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein, or an engineered immune cell disclosed herein, or the second pharmaceutical composition, for the manufacture of a medicament for inducing an immune response against a tumor having a TP53R282W mutation in a subject, and / or for preventing or treating a tumor having a TP53R282W mutation in a subject.

[0218] Advantages of the present application

[0219] Currently, TP53R282W gene mutation accounts for about 1% of human solid tumors, and according to the WHO statistics in 2022, there are 4.77 million new solid tumors in China every year, so about 47,000 solid tumor patients carry R282W mutation every year, and there is still no breakthrough drug. The present application provides epitope peptides of TP53R282W mutant, pMHC complexes and T cell receptors (TCRs) specifically recognizing the epitope peptides, cells and pharmaceutical compositions comprising the epitope peptides, pMHC complexes or TCRs, nucleic acids encoding the epitope peptides, pMHC complexes or TCRs, vectors and host cells for preparing the epitope peptides, pMHC complexes or TCRs, and methods for treating subjects using the epitope peptides, pMHC complexes or TCRs. The epitope peptides and TCRs provided by the present application can induce an immune response against tumors containing TP53R282W mutation and thus treat the above-mentioned tumors in subjects. In addition, the epitope peptides and TCRs provided by the present application are MHC-I restricted, which is the dominant allele in the Asian population, and thus are particularly suitable for patients in the Asian region. HLA-B5701 and B5801 have a distribution frequency of 30% in the Asian and southern Chinese ethnic groups and about 15% in the northern Chinese ethnic group, and belong to HLA with a relatively high frequency, so the drug can be used for a wider population in the future. Therefore, the present application provides a new T cell-based immunotherapy for the treatment of TP53R282W mutation-positive tumors, which has great clinical value.

[0220] The present application will be further clarified by the following examples, which should not be construed as limiting the scope of the application. Examples

[0221] Example 1. Tumor infiltrating T cell culture and expansion

[0222] The tumor resected from a pancreatic cancer patient with TP53 mutation R282W was cut into 2-4 mm tumor pieces with a surgical knife and washed twice with DPBS solution, and cultured in TIL medium containing IL2 (6000 IU / ml), human AB serum (2%), Hepes (25 mM), Xvivo15. Every 2-3 days, half of the medium was replaced, and when the tumor infiltrating T lymphocytes (TIL, Tumor infiltrating T Lymphocytes) grew to 60-80%, the TIL was harvested and frozen. The TIL cells were mixed and cultured in a T175 culture flask at a ratio of 1:30-1:200 with gamma-irradiated pooled donor-derived peripheral blood mononuclear cells (pooled PBMC, donors ≥3), and the cell number in each culture flask was not more than 1×10 8(10 ng / ml OKT3 added TIL medium), after 3 days of culture, half of the medium was replaced (Xvivo 15 medium with 3000 IU / ml IL2, 2% human AB serum), after 7 days of culture, cells were washed once and the medium was replaced and passaged at 1.0 x 10 6 / ml; cells were harvested after 10-14 days of culture for freezing.

[0223] Example 2. Preparation of TP53R282W antigen mRNA

[0224] The sequence containing TP53 mutation R282W was designed as follows to construct the vector UTR-LAMP3Lumenal-TP53 R282W -LMP3Sorting-UTR, the sequence is shown as SEQ ID NO: 38, wherein TP53 is marked with single underline and R282W mutation is marked with double underline (the nucleotide sequence of TP53-R282W mRNA is shown as SEQ ID NO: 37). The above sequence was synthesized by gene and cloned into pcDNA3.1 vector, mRNA was transcribed in vitro using T7 promoter (mMESSAGE mMACHINE T7 Transcription Kit, Thermofisher), and the transcribed mRNA was aliquoted and stored at -80°C.

[0225] Example 3. Screening of TP53R282W specific T cells

[0226] After the TIL cells were recovered, they were cultured for at least 48 hours for use in the screening experiment. The patient's autologous DC cells or LCL cells as antigen presenting cells (APCs) were used for TP53R282W mRNA transfection using a Neon electroporator, and the next day they were used for the screening experiment. In a 96-well U-bottom plate, 1-2 x 10 5 TIL cells and 0.5 x 10 5 DC cells or 4 x 10 5 LCL cells were cultured in Xvivo 15 medium, and after 16 hours of culture, the cell culture supernatant was collected, and the release of IFNγ in the supernatant was determined using Human IFNγ Flex Set, and the results of TIL screening are shown in Figure 1.

[0227] The results show that the TIL numbered R85.15 can specifically recognize the TP53R282W antigen peptide presented by APC cells.

[0228] Example 4. TCR sequencing and antigen specificity determination

[0229] TIL cells stimulated by APC cells were subjected to flow sorting using T cell activation specific markers (e.g. CD3 and CD137), TCR single cell sequencing was performed to obtain the TCR sequence of each cell.

[0230] TCR clones in single cell sequencing were gene synthesized in the order of TRAV-mCa-P2A-TRBV-mCb, wherein TRAV is the variable region of the alpha chain of the TCR, mCa is the murine TCR alpha constant region (the amino acid sequence and nucleotide sequence thereof are shown in SEQ ID NO: 2 and 3, respectively), TRBV is the variable region of the beta chain of the TCR, mCb is the murine TCR beta constant region (the amino acid sequence and nucleotide sequence thereof are shown in SEQ ID NO: 4 and 5, respectively), and P2A is a self-cleavage peptide (the nucleotide sequence thereof is shown in SEQ ID NO: 6); the above sequence was cloned into a lentiviral shuttle vector; the shuttle vector was transiently transfected into 293T cells according to the standard lentiviral vector packaging method, and the culture supernatant was collected, so that the lentiviral vector expressing the TCR was contained in the culture supernatant. T cells from a healthy donor were activated by OKT3 / 15E8 antibody-coated 6-well plates for 24 hours, transduced with lentiviral vectors containing TCR, and continued to be cultured for 6-8 days, and then subjected to TCR antigen-specific screening (antibodies recognizing murine TCR alpha or beta constant region were added for staining to detect the expression of recombinant TCR) to obtain T cells modified by recombinant TCR.

[0231] Autologous APC cells from patient No. R85 were loaded with TP53R282W long peptide (FEVRVCACPGRDWRTEEENLRKKGE, SEQ ID NO: 7) or TP53WT long peptide (FEVRVCACPGRDRRTEEENLRKKGE, SEQ ID NO: 8) and cultured overnight; the T cells modified by the recombinant TCR were inoculated with the peptide-loaded APC cells at a ratio of 1:1 in a 96-well U-bottom plate and cultured overnight, and the specific release of IFNγ in the supernatant was detected, with the culture medium as a blank control. The results are shown in Figure 2. The results show that the TCR clone No. R85.15.1 can specifically recognize the APC-presented TP53R282W mutant antigen peptide but not the wild-type TP53 peptide.

[0232] The sequence of R85.15.1 TCR is shown in Table 1.

[0233] Table 1. TCR sequence

[0234] Example 5. HLA restriction assay

[0235] Seven antigen presenting cells (LCL cell lines derived from different donors, LCLB9, LCLB11, LCLR85, LCLWQ, LCL076, LCL186) were loaded with TP53R282W long peptide (SEQ ID NO: 7) or TP53WT long peptide (SEQ ID NO: 8) respectively, and co-cultured with R85.15.1 TCR-T cells respectively, to determine the HLA restriction of R85.15.1 TCR by detecting IFNγ in the culture supernatant, and the results are shown in Figure 3.

[0236] LCL cell lines with HLA-B genotype of HLA-B*5701 (LCLB11 and LCL076) and HLA-B*5801 (LCLR85) can all present TP53R282W mutation recognized by R85.15.1 TCR-T cells, so the HLA restriction of R85.15.1 TCR is HLA-B*5701 and HLA-B*5801.

[0237] Table 2.

[0238] Example 6. Determination of presentation of TP53R282W on HLA-B*5801

[0239] HLA-B*5801 presentation of TP53R282W was determined using human pancreatic adenocarcinoma cells (AsPC1), human chronic myeloid leukemia cells (K562) and human osteosarcoma cells (U2OS). The three cell lines were transfected to overexpress HLA-B*5801 and / or TP53R282W respectively. Cells overexpressing only HLA-B*5801 were used as a control group, and cells co-overexpressing HLA-B*5801 and TP53R282W were used as an experimental group. The above cell lines were collected and resuspended in RPMI1640 medium respectively, and co-cultured with R85.15.1 TCR-T cells at a ratio of 2x10 4 :2x10 4 in RPMI1640 medium containing 2% fetal bovine serum, and the release of IFNγ in the supernatant was determined, and the results are shown in Figure 4.

[0240] The results show that TP53R282W mutation can be presented on HLA-B*5801 by tumor cell lines of different tissue origins, and R85.15.1 TCR can specifically recognize TP53R282W antigen peptide presented on HLA-B*5801.

[0241] Example 7. Determination of TP53R282W antigen peptide epitope

[0242] After the synthesized 8-25 amino acid peptides containing TP53 mutation R282W site (SEQ ID NO: 7, 22-36) were loaded into the autologous LCL cells (LCLR85) of patient No. R85 respectively, the antigen presenting cells were co-cultured with R82.15.1 TCR-T cells overnight at a ratio of 2x10 4 :2x10 4 The specific IFNγ release in the supernatant was determined, and the results are shown in Figure 5.

[0243] The results show that R282W antigen peptides, R282W-P1 and R282W-P15 can effectively induce IFNγ release of R85.15.1 TCR-T, indicating that R85.15.1 TCR can specifically recognize R282W-P1 (11mer), R282W-P15 (10mer) and the control 25mer antigen peptide presented by HLA-B*5801.

[0244] Table 3. TP53 R282W epitope screening sequences

[0245] Example 8. R85.15.1 TCR-T avidity determination

[0246] The TP53 R282W-P15 peptide segment (SEQ ID NO: 36) or its corresponding wild-type peptide segment (RVCACPGRDR) was loaded into the antigen presenting cells at a concentration of 10 ug / ml, 1 ug / ml, 0.1 ug / ml, 0.01 ug / ml or 0.001 ug / ml, and after incubation at 37°C for 2 hours, the R85.15.1 TCR-T cells were sorted by CD4 / 8 magnetic beads, and the APC cells and R85.15.1 TCR-T cells prepared in Example 4 were co-cultured overnight at a ratio of 2x10 4 :2x10 4 The IFNγ release in the supernatant was determined, and the results are shown in Figure 6.

[0247] The results show that the EC50 of R85.15.1 TCR recognizing TP53 R282W antigen peptide is 1.7 ug / ml.

[0248] Meanwhile, R85.15.1 TCR-T cells were prepared using CD4 or CD8 T cells to determine the CD4 / 8 dependence of their binding to pHLA complex. R85.15.1 TCR-CD4 T or R85.15.1 TCR-CD8 T cells were prepared according to the method of Example 4, and the supernatant was measured for IFNγ release after overnight incubation of APC cells and CD4 T or CD8 T cells. The results are shown in Figure 7. The calculated affinity of TCR-T to recognize TP53R282W antigen peptide is shown in Table 4. The results show that R85.15.1 TCR binding to pHLA (complex of HLA-B*5801 and TP53R282W-P15) has a certain dependence on CD8 T cell co-receptors, showing a higher affinity to CD8 T cells.

[0249] Table 4.

[0250] Sequence summary

[0251] Information on some of the sequences involved in the present application is provided in the following table.

[0252] Those skilled in the art will further appreciate that the application can be practiced in other specific forms without departing from its spirit or central characteristics. The foregoing description of the application has been disclosed for illustrative purposes, and it is understood that other variations are considered within the scope of the application. Therefore, the application is not limited to the specific embodiments described herein. Rather, it should be understood to be indicated by the appended claims.

Claims

1. An isolated epitope peptide consisting of 10-25 contiguous amino acid residues of a TP53 protein mutant having a R282W substitution, and comprising amino acid residues 273-282 of the TP53 protein mutant.

2. The epitope peptide of claim 1, wherein the epitope peptide is capable of being presented by an MHC class I molecule, and associates with an MHC class I molecule to form an MHC-peptide (pMHC) complex, which is capable of being recognized by a T cell, e.g., by an antigen-specific T cell receptor on a T cell; preferably, the MHC class I molecule in the pMHC complex is HLA-B; preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801.

3. The epitope peptide of claim 1 or 2, wherein the amino acid residues 273-282 of the TP53 protein mutant have a sequence as set forth in SEQ ID NO:

36.

4. The epitope peptide of any one of claims 1-3, wherein the epitope peptide comprises amino acid residues 273-283 or amino acid residues 273-282; preferably, the amino acid residues 273-283 or amino acid residues 273-282 of the TP53 protein mutant have a sequence as set forth in SEQ ID NO: 22 or 36, respectively.

5. The epitope peptide of any one of claims 1-4, wherein the TP53 protein mutant has a sequence as set forth in SEQ ID NO:

1.

6. A peptide-MHC (pMHC) complex comprising the epitope peptide of any one of claims 1-5 and an MHC molecule; preferably, the MHC molecule is an MHC class I molecule; preferably, the MHC class I molecule is HLA-B; preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801.

7. The pMHC complex of claim 6, wherein the epitope peptide is covalently linked to or associated with the MHC molecule by non-covalent interaction.

8. The pMHC complex of claim 6 or 7, which is in a soluble form or presented on a cell surface.

9. The pMHC complex of any one of claims 6-8, which is linked to other moieties selected from a therapeutic moiety, a fluorescent or luminescent label, a radioactive label, a nucleic acid probe and a contrast agent, an antibody, or an enzyme that generates a detectable product.

10. An isolated T cell receptor (TCR) or an antigen-binding fragment thereof, which is capable of specifically recognizing the epitope peptide of any one of claims 1-5 or the pMHC complex of any one of claims 6-9; preferably, the epitope peptide is presented by an MHC class I molecule; preferably, the MHC class I molecule is HLA-B; preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801; preferably, the TCR is soluble or membrane-bound. Preferably, the TCR is a full-length TCR, a soluble TCR, or a single-chain TCR.

11. An isolated T cell receptor (TCR) or antigen-binding fragment thereof that is capable of specifically recognizing a TP53 protein mutant having a R282W substitution, the TCR or antigen-binding fragment thereof comprising an alpha chain variable region (Va) and / or a beta chain variable region (Vp), wherein: (a) the Va comprises a CDR1a, a CDR2a, and a CDR3a; and / or (b) the Vp comprises a CDR1p, a CDR2p, and a CDR3p; Preferably, the TCR is soluble or membrane-bound. Preferably, the TCR is a full-length TCR, a soluble TCR, or a single-chain TCR.

12. The TCR or antigen-binding fragment thereof of claim 11, wherein: (i) the CDR1a comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 14; (ii) the CDR2a comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 15; (iii) the CDR3a comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

16.

13. The TCR or antigen-binding fragment thereof of claim 11, wherein: (i) the CDR1p comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 18; (ii) the CDR2p comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 19; (iii) the CDR3p comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

20.

14. The TCR or antigen-binding fragment thereof of any one of claims 11-13, wherein: (a) the Va comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 10; and / or (b) the Vp comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

12.

15. The TCR or antigen-binding fragment thereof according to any one of claims 11-14, wherein the TCR or antigen-binding fragment thereof is capable of specifically recognizing the epitope peptide according to any one of claims 1-5 or the pMHC complex of any one of claims 6-9; Preferably, the epitope peptide is presented by an MHC class I molecule; Preferably, the MHC class I molecule is HLA-B; Preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801; Preferably, when the TCR or antigen-binding fragment thereof is expressed on the surface of a T cell, the T cell is activated upon co-culturing with a second cell (e.g. an antigen presenting cell) displaying the epitope peptide according to any one of claims 1-5.

16. A conjugate comprising the TCR or antigen-binding fragment thereof according to any one of claims 10-15 and an effector moiety conjugated thereto; Preferably, the TCR or antigen-binding fragment thereof is soluble.

17. A fusion protein comprising the TCR or antigen-binding fragment thereof according to any one of claims 10-15 and a further peptide or protein; Preferably, the further peptide or protein comprises an anti-CD3 single chain antibody, IL-2 and mutants thereof, human immunoglobulin constant regions, microtubulin inhibitor conjugates, etc. Preferably, the TCR or antigen-binding fragment thereof is soluble.

18. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the epitope peptide according to any one of claims 1-5, or one or more nucleotide sequences encoding the epitope peptide and the MHC molecule of the pMHC complex according to any one of claims 6-9, or a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or the variable region of the alpha chain and / or the variable region of the beta chain according to any one of claims 10-15, or a nucleotide sequence encoding the fusion protein according to claim 17.

19. A vector comprising the nucleic acid molecule according to claim 18; Preferably, the vector is a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a baculoviral vector.

20. A host cell comprising the nucleic acid molecule according to claim 18 or the vector according to claim 19; Preferably, the host cell comprises an E. coli, a yeast, an insect cell, or a mammalian cell.

21. A method of producing the epitope peptide according to any one of claims 1-5, or the pMHC complex according to any one of claims 6-9, or the TCR or antigen-binding fragment thereof according to any one of claims 10-15, or the fusion protein according to claim 17, comprising: culturing the host cell according to claim 20 under conditions permitting protein expression, and recovering the epitope peptide, or the pMHC complex, or the TCR or antigen-binding fragment thereof, or the fusion protein from the culture of the host cell.

22. An engineered antigen presenting cell (APC) presenting on the cell surface an epitope peptide according to any one of claims 1-5 or a pMHC complex of any one of claims 6-9; Preferably, the epitope peptide is presented by an MHC class I molecule; Preferably, the MHC class I molecule is HLA-B; Preferably, the HLA-B comprises HLA-B*5701 and / or HLA-B*5801; Preferably, the APC is selected from a dendritic cell, a monocyte, a macrophage, a lymphoblast cell line, or any combination thereof; Preferably, the APC is HLA-B*5701 positive or HLA-B*5801 positive; Preferably, the APC is isolated from an HLA-B*5701 positive subject or an HLA-B*5801 positive subject.

23. A method of preparing the engineered APC of claim 22, comprising: (a) providing an APC from a subject; (b) contacting the APC in vitro with an epitope peptide according to any one of claims 1-5 or a pMHC complex of any one of claims 6-9 or introducing into the APC an expression vector comprising a nucleotide sequence encoding an epitope peptide according to any one of claims 1-5 or a pMHC complex of any one of claims 6-9, to obtain an APC presenting on the surface thereof the epitope peptide or the pMHC complex.

24. An engineered immune cell expressing on the cell surface a TCR or an antigen binding fragment thereof according to any one of claims 10-15; Preferably, the engineered immune cell comprises a nucleotide sequence encoding a TCR or an antigen binding fragment thereof according to any one of claims 10-15; Preferably, the immune cell is a lymphocyte; Preferably, the immune cell is selected from a T cell (e.g., an alpha beta T cell, a gamma delta T cell, or an iPSC-induced T cell), a tumor infiltrating lymphocyte (TIL), a natural killer (NK) cell, a natural killer T (NKT) cell, or any combination thereof.

25. A method of preparing the engineered immune cell of claim 24, comprising: (a) providing an immune cell from a subject; (b) introducing into the immune cell of step (a) the isolated nucleic acid molecule of claim 18 or the vector of claim 19, which comprises a nucleotide sequence encoding a TCR or an antigen binding fragment thereof according to any one of claims 10-15, to obtain an immune cell expressing the TCR or the antigen binding fragment thereof; Preferably, in step (a), the immune cell is pre-treated; the pre-treatment comprises sorting, activating, and / or proliferating the immune cell; Preferably, the pre-treatment comprises contacting the immune cell with an anti-CD3 antibody and / or an anti-CD28 antibody, thereby stimulating the immune cell and inducing its proliferation, thereby generating the pre-treated immune cell.

26. A pharmaceutical composition comprising an epitope peptide according to any one of claims 1-5, a pMHC complex of any one of claims 6-9, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the epitope peptide or pMHC complex, or an engineered antigen presenting cell (APC) according to claim 22; and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition is a tumor vaccine. Preferably, the pharmaceutical composition comprises an adjuvant. Preferably, the pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immune-enhancing agent.

27. The pharmaceutical composition of claim 26, wherein the anti-tumor agent is selected from an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an anti-metabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, or a TIM3 antibody).

28. The pharmaceutical composition of claim 26, wherein the immune-enhancing agent is selected from an immune-stimulatory antibody (e.g., an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD40L (CD154) antibody, an anti-41BB (CD137) antibody, an anti-OX40 antibody, an anti-GITR antibody, or any combination thereof) or an immune-stimulatory cytokine (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-gamma, IL-10, TGF-beta, GM-CSF, or any combination thereof).

29. A pharmaceutical composition comprising a TCR or antigen-binding fragment thereof according to any one of claims 10-15, a conjugate according to claim 16, a fusion protein according to claim 17, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein, or an engineered immune cell according to claim 24; and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immune-enhancing agent.

30. The pharmaceutical composition of claim 29, wherein the anti-tumor agent is selected from an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an anti-metabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, or a TIM3 antibody).

31. The pharmaceutical composition of claim 29, wherein the immune-enhancing agent is selected from an immunostimulatory antibody (e.g., an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD40L (CD154) antibody, an anti-41BB (CD137) antibody, an anti-OX40 antibody, an anti-GITR antibody, or any combination thereof) or an immunostimulatory cytokine (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-g, IL-10, TGF-b, GM-CSF, or any combination thereof).

32. Use of an epitope peptide according to any one of claims 1-5, a pMHC complex according to any one of claims 6-9, a nucleic acid molecule or a vector or a host cell comprising a nucleotide sequence encoding said epitope peptide or pMHC complex, or an engineered antigen presenting cell (APC) according to claim 22, or a pharmaceutical composition according to any one of claims 26-28, for the manufacture of a medicament for inducing an immune response against a tumor having a TP53 R282W mutation in a subject, and / or for preventing or treating a tumor having a TP53 R282W mutation in a subject; preferably, the tumor having a TP53 R282W mutation is selected from uterine cancer, ovarian cancer, head and neck cancer, small cell lung cancer, pancreatic cancer, lung adenocarcinoma, and non-small cell lung cancer; preferably, the subject is a human; preferably, the subject is HLA-B*5701 positive or HLA-B*5801 positive; preferably, the epitope peptide, nucleic acid molecule or vector or host cell, engineered antigen presenting cell (APC), or pharmaceutical composition is administered in combination with, e.g., simultaneously, separately, or sequentially, an additional therapeutic agent; preferably, the additional therapeutic agent is an immunostimulatory agent or an anti-tumor agent.

33. Use of a TCR or antigen-binding fragment thereof according to any one of claims 10-15, a conjugate according to claim 16, a fusion protein according to claim 17, a nucleic acid molecule or a vector or a host cell comprising a nucleotide sequence encoding said TCR or antigen-binding fragment thereof or fusion protein, or an engineered immune cell according to claim 24, or a pharmaceutical composition according to any one of claims 29-31, for the manufacture of a medicament for inducing an immune response against a tumor having a TP53 R282W mutation in a subject, and / or for preventing or treating a tumor having a TP53 R282W mutation in a subject; wherein the nucleic acid molecule, vector or host cell comprises a nucleotide sequence encoding said TCR or antigen-binding fragment thereof or fusion protein; preferably, the tumor having a TP53 R282W mutation is selected from uterine cancer, ovarian cancer, head and neck cancer, small cell lung cancer, pancreatic cancer, lung adenocarcinoma, and non-small cell lung cancer; preferably, the subject is a human; preferably, the subject is HLA-B*5701 positive or HLA-B*5801 positive; Preferably, the TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule or vector or host cell, engineered immune cell, or pharmaceutical composition is administered in combination with, e.g. simultaneously, separately or sequentially, another therapeutic agent; preferably the other therapeutic agent is an immunostimulatory agent or an anti-tumour agent.

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