T cell receptors and methods of use thereof

Abstract

This disclosure relates to a recombinant T-cell receptor (and fragment thereof) capable of binding to the HPV16 E6 epitope and a nucleic acid molecule encoding said recombinant T-cell receptor (and fragment thereof). In some aspects, the nucleic acid molecule further comprises a second nucleotide sequence, wherein said second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the expression of endogenous TCRs. Other aspects of this disclosure relate to vectors and cells, said vectors comprising said nucleic acid molecules, said cells comprising said recombinant TCRs, said nucleic acid molecules, or said vectors. Still other aspects of this disclosure relate to methods of using said recombinant T-cell receptor (and fragment thereof). In some aspects, said methods include treating a subject with cancer in need.

Description

[0001] Cross-references to related applications This PCT application claims priority to U.S. Provisional Application No. 63 / 592,842, filed October 24, 2023, which is incorporated herein by reference in its entirety.

[0002] References to sequence lists submitted electronically The contents of the sequence list submitted electronically (filename: 4706_021PC01_SequenceListing_ST26.xml; size: 149,442 bytes; creation date: October 23, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure provides a recombinant T-cell receptor (“TCR”) that specifically binds to human papillomavirus (HPV) transforming protein E6 (HPV16 E6) and its uses. Background Technology

[0004] Immunotherapy has become a key tool in the fight against a variety of diseases, including cancer. T-cell therapy is at the forefront of immunotherapy development, and adoptive transfer of anti-tumor T cells has been shown to induce clinical responses in cancer patients. Although many T-cell therapies target mutated tumor antigens, the vast majority of these neoantigens are not shared and are unique to each patient.

[0005] The number of potential non-mutated antigens is orders of magnitude greater than that of mutated antigens. Elucidating T-cell epitopes derived from shared antigens could contribute to the robust development of effective and safe adoptive T-cell therapies, which could be readily applied to a larger population of cancer patients. However, the absolute number of non-mutated antigens and the high polymorphism of HLA genes may hinder a comprehensive analysis of the specificity of anti-tumor T-cell responses to non-mutated antigens. Summary of the Invention

[0006] Some aspects of this disclosure relate to a nucleic acid molecule comprising a nucleotide sequence encoding a recombinant T-cell receptor (TCR) or its antigen-binding portion (“anti-HPV16 E6TCR”) that specifically binds to human papillomavirus (HPV) converting protein E6 (HPV16 E6), wherein the anti-HPV16 E6 TCR cross-competes with a reference TCR comprising an α-chain and a β-chain for binding to human HPV16 E6, and wherein the α-chain comprises an amino acid sequence as shown in SEQ ID NO: 1, and the β-chain comprises an amino acid sequence as shown in SEQ ID NO: 2. Some aspects of this disclosure relate to a nucleic acid molecule comprising a nucleotide sequence encoding a recombinant T-cell receptor (TCR) that specifically binds to human HPV16 E6 or its antigen-binding portion (“anti-HPV16 E6 TCR”), wherein the anti-HPV16 E6 TCR binds an epitope or overlapping epitope of human HPV16 E6 identical to that of a reference TCR comprising an α-chain and a β-chain, wherein the α-chain comprises an amino acid sequence as shown in SEQ ID NO: 1, and the β-chain comprises an amino acid sequence as shown in SEQ ID NO: 2. In some aspects, the nucleic acid molecule further comprises a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the expression of endogenous TCRs.

[0007] Some aspects of this disclosure relate to a nucleic acid molecule comprising: (i) a first nucleotide sequence encoding a recombinant T-cell receptor (TCR) or its antigen-binding portion (“anti-HPV16 E6 TCR”) that specifically binds to human papillomavirus (HPV) converting protein E6 (HPV16 E6); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR, wherein the anti-HPV16 E6 TCR cross-competes with a reference TCR comprising an α chain and a β chain for binding to human HPV16 E6, and wherein the α chain comprises an amino acid sequence as shown in SEQ ID NO: 1, and the β chain comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0008] Some aspects of this disclosure relate to a nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T-cell receptor (TCR) or its antigen-binding portion (“anti-HPV16 E6TCR”) that specifically binds to human HPV16 E6; and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR, wherein the anti-HPV16 E6 TCR binds an epitope or overlapping epitope of human HPV16 E6 that is identical to a reference TCR comprising an α-chain and a β-chain, wherein the α-chain comprises an amino acid sequence as shown in SEQ ID NO: 1, and the β-chain comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0009] In some respects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6, which consists of an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

[0010] In some respects, epitopes are complexed with HLA class I molecules. In some respects, HLA class I molecules are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G alleles. In some respects, HLA class I molecules are the HLA-C*07 allele. In some respects, HLA class I molecules are the HLA-C*07:01 or HLA-C*07:02 alleles.

[0011] In some respects, the anti-HPV16 E6 TCR comprises an α chain and a β chain, wherein the α chain contains variable regions comprising α chain CDR1, α chain CDR2 and α chain CDR3; and wherein the β chain contains variable regions comprising β chain CDR1, β chain CDR2 and β chain CDR3; wherein the α chain CDR3 contains an amino acid sequence as shown in SEQ ID NO: 7.

[0012] In some aspects, the β-chain CDR3 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the anti-HPV16 E6 TCR comprises an α-chain and a β-chain, wherein the α-chain comprises a variable region containing α-chain CDR1, α-chain CDR2, and α-chain CDR3; and wherein the β-chain comprises a variable region containing β-chain CDR1, β-chain CDR2, and β-chain CDR3; wherein the β-chain CDR3 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO: 10.

[0013] In some aspects, the α-chain CDR3 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the α-chain CDR1 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 5. In some aspects, the β-chain CDR1 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the α-chain CDR2 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the β-chain CDR2 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 9.

[0014] In some aspects, the α-chain variable region of the anti-HPV16 E6 TCR contains the amino acid sequence of the variable region present in the amino acid sequence shown in SEQ ID NO: 1. In some aspects, the β-chain variable region of the anti-HPV16 E6 TCR contains the amino acid sequence of the variable region present in the amino acid sequence shown in SEQ ID NO: 2.

[0015] In some aspects, the α-chain of the anti-HPV16 E6 TCR also includes a constant region, wherein this constant region is different from the endogenous constant region of the α-chain. In some aspects, the α-chain constant region of the anti-HPV16 E6 TCR also includes a constant region, wherein the α-chain constant region contains an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region present in the amino acid sequence shown in SEQ ID NO: 1. In some aspects, the α-chain constant region contains an amino acid sequence comprising at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO: 1.

[0016] In some aspects, the β-chain of the anti-HPV16 E6 TCR also includes a constant region, wherein this constant region is different from the endogenous constant region of the β-chain. In some aspects, the β-chain constant region of the anti-HPV16 E6 TCR also includes a constant region, wherein the β-chain constant region contains an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region present in the amino acid sequence shown in SEQ ID NO: 2. In some aspects, the β-chain constant region contains an amino acid sequence comprising at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO: 2.

[0017] In some aspects, the α chain of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 1. In some aspects, the β chain of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 2.

[0018] In some respects, the second nucleotide sequence is one or more siRNAs that reduce the expression of endogenous TCRs. In some respects, one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the endogenous TCR. In some respects, one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 25-28.

[0019] In some respects, the anti-HPV16 E6 TCR comprises an α-chain constant region, a β-chain constant region, or both thereof; and wherein the α-chain constant region, the β-chain constant region, or both thereof comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five substitutions within the target sequence relative to the corresponding amino acid sequence of the endogenous TCR.

[0020] In some aspects, the α chain contains a signal peptide, the β chain contains a signal peptide, or both the α chain and the β chain contain a signal peptide. In some aspects, the signal peptide contains an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 20-22 and any combination thereof.

[0021] Some aspects of this disclosure relate to vectors comprising the nucleic acid molecules disclosed herein. In some aspects, the vector is a viral vector, a mammalian vector, or a bacterial vector. In some aspects, the vector is a retroviral vector. In some aspects, the vector is selected from the group consisting of: adenovirus vectors, lentiviruses, Sendai virus vectors, baculovirus vectors, Epstein-Barr virus vectors, papillomatosis vacuolating virus vectors, vaccinia virus vectors, herpes simplex virus vectors, mixed vectors, or adeno-associated virus (AAV) vectors. In some aspects, the vector is a lentivirus.

[0022] Some aspects of this disclosure relate to a T-cell receptor (TCR) or its antigen-binding portion, which includes the α-chain variable region of the anti-HPV16 E6 TCR disclosed herein and the β-chain variable region of the anti-HPV16 E6 TCR disclosed herein.

[0023] Some aspects of this disclosure relate to a recombinant T-cell receptor (TCR) or its antigen-binding portion (“anti-HPV16 E6 TCR”) that specifically binds to human HPV16 E6, which cross-competes with a reference TCR for binding to human HPV16 E6; wherein the reference TCR comprises an α chain and a β chain, and wherein the α chain comprises an amino acid sequence as shown in SEQ ID NO: 1, and the β chain comprises an amino acid sequence as shown in SEQ ID NO: 2; and wherein the anti-HPV16 E6 TCR comprises an α chain and a β chain, wherein the α chain comprises a constant region, and wherein the β chain comprises a constant region; wherein (i) the α chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO: 1, or (ii) the β chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO: 2.

[0024] Some aspects of this disclosure relate to a recombinant T-cell receptor (TCR) or its antigen-binding portion (“anti-HPV16 E6 TCR”) that specifically binds to human HPV16 E6 epitopes or overlapping epitopes of human HPV16 E6 as in a reference TCR; wherein the reference TCR comprises an α chain and a β chain, and wherein the α chain comprises an amino acid sequence as shown in SEQ ID NO: 1, and the β chain comprises an amino acid sequence as shown in SEQ ID NO: 2; and wherein the anti-HPV16 E6 TCR comprises an α chain and a β chain, wherein the α chain comprises a constant region, and wherein the β chain comprises a constant region; wherein (i) the α chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO: 1, or (ii) the β chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO: 2. In some respects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6, which consists of an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

[0025] In some respects, epitopes are complexed with HLA class I molecules. In some respects, HLA class I molecules are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G alleles. In some respects, HLA class I molecules are the HLA-C*07 allele. In some respects, HLA class I molecules are the HLA-C*07:01 or HLA-C*07:02 alleles.

[0026] In some respects, the α chain of the anti-HPV16 E6 TCR includes variable regions containing α chain CDR1, α chain CDR2 and α chain CDR3; and the β chain of the anti-HPV16 E6 TCR includes variable regions containing β chain CDR1, β chain CDR2 and β chain CDR3; wherein the α chain CDR3 of the anti-HPV16 E6 contains an amino acid sequence as shown in SEQ ID NO: 7.

[0027] In some aspects, the β-chain CDR3 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the α-chain of the anti-HPV16 E6 TCR contains variable regions containing α-chain CDR1, α-chain CDR2, and α-chain CDR3; wherein the β-chain of the anti-HPV16 E6 TCR contains variable regions containing β-chain CDR1, β-chain CDR2, and β-chain CDR3; and wherein the β-chain CDR3 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 10.

[0028] In some aspects, the α-chain CDR3 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the α-chain CDR1 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 5. In some aspects, the β-chain CDR1 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the α-chain CDR2 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the β-chain CDR2 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 9.

[0029] In some aspects, the α-chain variable region of the anti-HPV16 E6 TCR contains the amino acid sequence of the variable region present in the amino acid sequence shown in SEQ ID NO: 1. In some aspects, the β-chain variable region of the anti-HPV16 E6 TCR contains the amino acid sequence of the variable region present in the amino acid sequence shown in SEQ ID NO: 2.

[0030] In some aspects, the α-chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence present in the constant region shown in SEQ ID NO: 1. In some aspects, the β-chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence present in the constant region shown in SEQ ID NO: 2.

[0031] In some aspects, the α chain of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 1. In some aspects, the β chain of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 2.

[0032] In some aspects, the α chain contains a signal peptide, the β chain contains a signal peptide, or both the α chain and the β chain contain a single peptide. In some aspects, the signal peptide contains an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 20-22 and any combination thereof.

[0033] Some aspects of this disclosure relate to a bispecific molecule comprising a first antigen-binding molecule and a second antigen-binding molecule, wherein the first antigen-binding molecule comprises a TCR disclosed herein or its antigen-binding portion. In some aspects, the first antigen-binding molecule comprises a single-stranded variable fragment (“scFv”).

[0034] In some respects, second antigen-binding molecules specifically bind to proteins expressed on the surface of T cells. In some respects, second antigen-binding molecules specifically bind to CD3. In some respects, second antigen-binding molecules contain scFv.

[0035] In some respects, the first antigen-binding molecule and the second antigen-binding molecule are linked or associated via covalent bonds. In other respects, the first antigen-binding molecule and the second antigen-binding molecule are linked via peptide bonds.

[0036] Some aspects of this disclosure relate to a cell comprising the nucleic acid molecules disclosed herein, the vectors disclosed herein, the TCRs disclosed herein, the recombinant TCRs disclosed herein, or the bispecific molecules disclosed herein. In some aspects, the cell further expresses CD3. In some aspects, the cell is selected from the group consisting of: T cells, natural killer (NK) cells, natural killer T (NKT) cells, or ILC cells.

[0037] Some aspects of this disclosure relate to a method of treating a subject with cancer, comprising administering to the subject the TCR disclosed herein, the bispecific molecule disclosed herein, the nucleic acid disclosed herein, or the cell disclosed herein. In some aspects, the cancer is selected from the group consisting of: HPV-positive head and neck cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer, or any combination thereof. In some aspects, the cancer is recurrent or refractory. In some aspects, the cancer is locally advanced. In some aspects, the cancer is advanced. In some aspects, the cancer is metastatic.

[0038] In some respects, cells are obtained from the subject. In other respects, cells are obtained from donors other than the subject.

[0039] In some respects, preconditioning is performed on the subject prior to administration of the cells. In some respects, preconditioning includes administering chemotherapy, cytokines, proteins, small molecules, or any combination thereof to the subject. In some respects, preconditioning includes administering interleukins. In some respects, preconditioning includes administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some respects, preconditioning includes administering a preconditioning agent selected from the group consisting of cyclophosphamide, fludarabine, vitamin C, AKT inhibitors, ATRA, rapamycin, or any combination thereof. In some respects, preconditioning includes administering cyclophosphamide, fludarabine, or both.

[0040] Some aspects of this disclosure relate to a method for engineering antigen-targeting cells, comprising transducing cells collected from a subject requiring T-cell therapy using nucleic acid molecules or vectors disclosed herein. In some aspects, the antigen-targeting cells further express CD3. In some aspects, the cells are T cells or natural killer (NK) cells.

[0041] Some aspects of this disclosure relate to an HLA class I molecule complexed with a peptide, wherein the HLA class I molecule comprises an α chain and a β chain; and wherein the peptide consists of an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

[0042] In some respects, HLA class I molecules are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G alleles. In other respects, HLA class I molecules are HLA-C alleles. In some respects, HLA class I molecules are HLA-C*07 alleles. In some respects, HLA class I molecules are HLA-C*07:01 or HLA-C*07:02 alleles.

[0043] In some respects, HLA class I molecules are monomers. In some respects, HLA class I molecules are dimers. In some respects, HLA class I molecules are trimers. In some respects, HLA class I molecules are tetramers. In some respects, HLA class I molecules are pentamers.

[0044] Some aspects of this disclosure relate to an antigen-presenting cell (APC) that contains the HLA class I molecules disclosed herein. In some aspects, the HLA class I molecules are expressed on the surface of the APC.

[0045] Some aspects of this disclosure relate to a method for enriching a population of target T cells obtained from a human subject, comprising contacting T cells with an HLA class I molecule disclosed herein or an APC disclosed herein, wherein, after contact, the enriched population of T cells contains a higher number of HLA class I-binding T cells relative to the number of T cells capable of binding HLA class I molecules prior to contact.

[0046] Some aspects of this disclosure relate to a method for enriching a population of target T cells obtained from a human subject, comprising contacting T cells in vitro with a peptide, wherein the peptide consists of an amino acid sequence as shown in SEQ ID NO: 13, wherein, after contact, the enriched population of T cells contains a higher number of tumor-targeting T cells relative to the number of tumor-targeting T cells prior to contact. In some aspects, the T cells obtained from the human subject are tumor-infiltrating lymphocytes (TILs).

[0047] Some aspects of this disclosure relate to an immunoconjugate comprising (i) the TCR or bispecific molecule disclosed herein and (ii) a drug. In some aspects, the drug includes a cytotoxic agent. In some aspects, the drug includes maytansinoid, dolastatin, duocarmycin, pyrrolobenzodiazepine, calicheamicin, amatoxin, or analogues thereof.

[0048] Some aspects of this disclosure relate to a method of treating a tumor in a subject of need, comprising administering to the subject an enriched population of T cells disclosed herein. Some aspects of this disclosure relate to a method of treating a tumor in a subject of need, comprising administering to the subject a TCR disclosed herein. Some aspects of this disclosure relate to a method of treating a tumor in a subject of need, comprising administering to the subject a bispecific molecule disclosed herein. Some aspects of this disclosure relate to a method of treating a tumor in a subject of need, comprising administering to the subject a nucleic acid disclosed herein. Some aspects of this disclosure relate to a method of treating a tumor in a subject of need, comprising administering to the subject an immune conjugate disclosed herein.

[0049] Some aspects of this disclosure relate to a method for enhancing cytotoxic T-cell-mediated cancer cell targeting in a subject with cancer, comprising administering to the subject a peptide having an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

[0050] Some aspects of this disclosure relate to a cancer vaccine comprising a peptide having an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

[0051] Some aspects of this disclosure relate to a method for selecting T cells capable of targeting tumor cells, comprising contacting an isolated population of T cells in vitro with a peptide, wherein the peptide consists of an amino acid sequence as shown in SEQ ID NO: 97, 102, 103, or 104. In some aspects, the T cells are tumor-infiltrating lymphocytes (TILs). Attached Figure Description

[0052] Figure 1A-1C It is for unstimulated TILs ( Figure 1A TILs stimulated with C*07:02 aAPC using a pulse of 20-meric peptide spanning the entire E6 protein () Figure 1B ) or TILs stimulated with anti-CD3 antibodies ( Figure 1C Graphical representation of flow cytometry data by TNF-α and CD8 expression. Figure 1D-1G This is a graphical representation of flow cytometry data of primary TILs stimulated with C*07:02 aAPC pulses containing the peptide 15 deletion (HPV-16 E6(53-61), AFRDLCIVY) and gated by TNFα and INFγ. Unstimulated and anti-CD3 stimulated TILs were used as negative and positive controls, respectively.

[0053] Figure 2This is a bar graph showing the peptide Ag-dependent IFNγ production of TILs stimulated by APCs presenting HLA-C*07:02 loaded with 29 individual E6 eicoseptides (Table 5).

[0054] Figure 3 It is a bar graph showing the production of TNFα and INFγ in primary tumor-infiltrating lymphocytes.

[0055] Figure 4 This is a bar graph showing IL-2 production in a human T cell line transduced with C*07:02 / HPV-16 E6 TCR.

[0056] Figure 5A-5T It is the expression of CD69 in human T cell lines that express α-β TCR cloned from primary human TIL. Figure 5A-5J ) and NFAT-GFP reporter gene signal ( Figure 5K-5T The graph represents the effect. Cells were stimulated with C*07:02aAPC pulsed with the entire HPV16 E6 protein, or with C*07:02aAPC pulsed with peptide 15 (HPV-16 E6(53-61), AFRDLCIVY; SEQ ID NO: 104). CD3-negative and PMA / ionomycin-stimulated T cells were used as negative and positive controls, respectively.

[0057] Figure 6A and 6B This demonstrates CD69 expression in a human T cell line that expresses α-β TCR from a primary human TIL clone. Figure 6A ) and NFAT-GFP reporter gene signal ( Figure 6B Bar graph of HPV16 E6 protein pulses. Cells were stimulated with C*07:02 aAPC pulses containing the entire HPV16 E6 protein, or with C*07:02 aAPC pulses containing the deletion of peptide 15 (HPV-16 E6(53-61), AFRDLCIVY; SEQ ID NO: 104). CD3-negative and PMA / ionomycin-stimulated T cells were used as negative and positive controls, respectively.

[0058] Figure 7 This is a graphical representation of the stimulation of primary TCR-T cells by HPV E6 deletion peptides, as measured by IFNγ secretion at various concentrations of deletion peptide 10 (SEQ ID NO: 99), deletion peptide 14 (SEQ ID NO: 103), and deletion peptide 15 (SEQ ID NO: 104).

[0059] Figures 8A-8F It is engineered to express C*07:02 / HPV-E6 (53-61) TCR ( Figure 8A ,8C (and 8E) or with unrelated TCR DPB1*04:01 / MAGE-A2 (108-127) ( Figure 8B and 8C (or as a negative control, A*02:01 / NYESO1) (157-165) ( Figure 8F Primary T cells () Figures 8A-8D ) and human T cell line ( Figure 8E-8F Graphical representation of multimer staining.

[0060] Figure 9 This is a schematic diagram of the HLA C*07:02:01 polypeptide.

[0061] Figure 10 It is 53 AFRDLCIVY 61 (SEQ ID NO: 104; missing peptide 15) Graphical representation of IFNγ production in C*07:02 / HPV-E6 and C*07:01 / HPV-E6 primary TCR-T cells stimulated by C*07:01 aAPC pulses.

[0062] Figure 11 This is a demonstration of C*07:02 / HPV-E6 after exposure to the HPV oncogenic protein E6. 53-61 Bar graph showing IFNγ production in T cells transduced by TCR. Detailed Implementation

[0063] This disclosure relates to a TCR or its antigen-binding portion thereof that specifically binds to an epitope on HPV16 E6, a nucleic acid molecule encoding the TCR or its antigen-binding portion thereof, and a cell containing the TCR or the nucleic acid molecule. Some aspects of this disclosure relate to methods of treating cancer in a subject of need. Other aspects of this disclosure relate to HLA class I molecules complexed with a peptide containing an epitope of HPV16 E6.

[0064] I. Terminology To make this disclosure more readily understandable, certain terms are first defined. As used in this application, each of the following terms shall have the meaning described below unless expressly provided otherwise herein. Further definitions are set forth throughout the application.

[0065] It should be noted that the term "a / an" refers to one or more of the entities described; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a," "one or more," and "at least one" are used interchangeably herein.

[0066] Furthermore, the term “and / or” as used herein should be considered as a specific disclosure of each of the two specified features or components having or not having the other. Thus, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0067] The term "about" is used in this document to mean approximately, roughly, or around. When used with a numerical range, it modifies the range by extending the boundaries above and below the value. Generally, the term "about" is used in this document to modify a value by a variation of about 10% above or below the value (increase or decrease).

[0068] It should be understood that whenever an aspect is described in this document as “comprising”, other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd edition, 2002, CRC Press; *Dictionary of Cell and Molecular Biology*, 3rd edition, 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, revised edition, 2000, Oxford University Press, provide a general dictionary for those skilled in the art of the use of many terms in this disclosure.

[0070] Units, prefixes, and symbols are represented in their accepted International System of Units (SI) forms. Numerical ranges include the values ​​defining the range. Unless otherwise indicated, nucleotide sequences are written from left to right with a 5' to 3' orientation. Amino acid sequences are written from left to right with an amino to carboxyl orientation. The headings provided herein are not intended to limit the various aspects of this disclosure, which are derived from the entire specification. Therefore, the terms defined below are more fully defined by reference to the entire specification.

[0071] "Administration" means the physical introduction of an agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a method of administration other than enteral and local administration, typically by injection, and including, but not limited to, intravenous, intramuscular, intraarterial, intrasheath, intralymphatic, intralesional, intracapsular, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. In some aspects, the formulation is administered via non-parenteral routes, such as oral administration. Other non-parenteral routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0072] As used herein, the term “T-cell receptor” (TCR) refers to a heteromeric cell surface receptor capable of specifically interacting with a target antigen. As used herein, “TCR” includes, but is not limited to, naturally occurring and non-naturally occurring TCRs; full-length TCRs and their antigen-binding portions; chimeric TCRs; TCR fusion constructs; and synthetic TCRs. In humans, TCRs are expressed on the surface of T cells, and they are responsible for T-cell recognition and targeting by antigen-presenting cells. Antigen-presenting cells (APCs) display fragments of exogenous proteins (antigens) that are complexed with the major histocompatibility complex (MHC, also referred to herein as complexed with HLA molecules (e.g., HLA class I molecules)). TCRs recognize and bind to the peptide:HLA complex and recruit CD8 (for MHC class I molecules) or CD4 (for MHC class II molecules), thereby activating the TCR. Activated TCRs initiate downstream signaling and immune responses, including the disruption of EPCs.

[0073] Typically, a TCR can consist of two chains, an α-chain and a β-chain (or less commonly, a γ-chain and a δ-chain), interconnected by disulfide bonds. Each chain contains variable regions (α-chain variable regions and β-chain variable regions) and constant regions (α-chain constant regions and β-chain constant regions). The variable regions are located distal to the cell membrane and interact with antigens. The constant regions are located proximal to the cell membrane. A TCR may further include a transmembrane region and a short cytoplasmic tail region. As used herein, the term "constant region" encompasses both the transmembrane region and the cytoplasmic tail region (when present), as well as the conventional "constant region".

[0074] The variable region can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), interspersed with more conserved regions (called framework regions (FRs)). Each α-chain and β-chain variable region contains three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Each variable region contains a binding domain that interacts with the antigen. Although all three CDRs on each chain are involved in antigen binding, CDR3 is considered the primary antigen-binding region, while CDR1 and CDR2 are considered to primarily recognize HLA molecules.

[0075] Unless otherwise specified, the term "TCR" also includes the antigen-binding portion of any TCR disclosed herein, and includes monovalent and bivalent fragments or portions, as well as single-chain TCRs. The term "TCR" is not limited to naturally occurring TCRs that bind to the surface of T cells. As used herein, the term "TCR" further refers to TCRs expressed on the surface of cells other than T cells (e.g., cells naturally expressing or modified to express CD3 as described herein) or TCRs without a cell membrane (e.g., isolated or soluble TCRs) as described herein.

[0076] "Antigen-binding fraction," "part of the TCR," or "TCR fragment" refers to any portion smaller than the entire TCR, which can be less than 1 × 10⁻⁶. 6 M of K D It specifically binds to the same antigen as the TCR. The antigen-binding portion may include the antigen CDR.

[0077] “Antigen” refers to any molecule, such as a peptide, that elicits an immune response or can be bound by a TCR. As used herein, “epitope” refers to a portion of a polypeptide that elicits an immune response or can be bound by a TCR. An immune response may involve the production of antibodies or the activation of specific immune-active cells, or both. Those skilled in the art will readily understand that any macromolecule (including virtually all proteins or peptides) can be used as an antigen. Antigens and / or epitopes can be expressed endogenously, i.e., through genomic DNA, or they can be recombinantly expressed. Antigens and / or epitopes may be specific to a particular tissue, such as cancer cells, or they may be widely expressed. Furthermore, fragments of larger molecules can act as antigens. In one respect, the antigen is a tumor antigen. Epitopes may be present in longer polypeptides (e.g., proteins), or epitopes may exist as fragments of longer polypeptides. In some respects, epitopes are complexed with the major histocompatibility complex (MHC) (also referred to herein as complexed with HLA molecules, such as HLA class 1 molecules).

[0078] As used herein, “HPV16 E6” or “human papillomavirus (HPV) transforming protein E6” refers to the human regulatory component of the cyclin D1-CDK4 (DC) complex, which phosphorylates and inhibits members of the retinoblastoma (RB) protein family (including RB1) and regulates the cell cycle during the G1 / S phase transition. Phosphorylation of RB1 allows the transcription factor E2F to dissociate from the RB / E2F complex and subsequently transcribe E2F target genes responsible for G1 phase progression. HPV16 E6 is also involved in the hypophosphorylation of RB1 in early G1 phase. The cyclin D-CDK4 complex is a major integrator of various pro-mitotic and anti-mitotic signals. HPV16 E6 is also a substrate of SMAD3, phosphorylating SMAD3 in a cell cycle-dependent manner and inhibiting its transcriptional activity. HPV16 E6 is also a component of the ternary complex cyclin D1 / CDK4 / CDKN1B, which is required for the nuclear translocation and activity of the cyclin D-CDK4 complex. Furthermore, HPV16 E6, along with INSM1, exhibits transcriptional co-repressor activity against the NEUROD1 and INS promoters in a cell cycle-independent manner. Mutations, amplifications, and overexpressions of HPV16 E6 that alter cell cycle progression are frequently observed in various tumors and may contribute to tumorigenesis.

[0079] As used herein, HPV16 E6 refers not only to the full-length canonical sequence but also to its variants and fragments. The amino acid sequence of HPV16 E6 (SEQ ID NO: 16) is provided in Table 1.

[0080] Table 1. HPV16 E6 amino acid sequence As used herein, the term "HLA" refers to human leukocyte antigens. In humans, HLA genes encode major histocompatibility complex (MHC) proteins. MHC proteins are expressed on the surface of cells and participate in the activation of immune responses. HLA class I genes encode MHC class I molecules, which are expressed on the surface of cells that have complexed with peptide fragments (antigens) of self or non-self proteins. T cells expressing TCR and CD3 recognize the antigen:MHC class I complex and initiate an immune response to target and destroy antigen-presenting cells displaying non-self proteins.

[0081] As used herein, "HLA class I molecule" or "HLA class I molecule" refers to the protein product of a wild-type or variant HLA class I gene encoding an MHC class I molecule. Therefore, "HLA class I molecule" and "MHC class I molecule" are used interchangeably in this article.

[0082] MHC class I molecules consist of two protein chains: an α chain and a β2-microglobulin (β2m) chain. Human β2m is encoded by the B2M gene. The amino acid sequence of β2m is shown in SEQ ID NO: 17 (Table 2). The α chain of MHC class I molecules is encoded by the HLA gene complex. The HLA complex is located in the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes with different functions. HLA genes are highly variable, with more than 20,000 HLA alleles and associated alleles, including more than 15,000 HLA class I alleles known in the art, which encode thousands of HLA proteins, including more than 10,000 HLA class I proteins (see, for example, hla.alleles.org, last accessed: February 27, 2019). At least three genes exist in the HLA complex encoding MHC class I α-chain proteins: HLA-A, HLA-B, and HLA-C. In addition, HLA-E, HLA-F, and HLA-G encode proteins that associate with MHC class I molecules.

[0083] Table 2. Amino acid sequence of human β2m The term "autologous" refers to any material derived from the same individual and subsequently reintroduced into that individual. For example, autologous T-cell therapy involves administering T cells isolated from the same individual to a subject. The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species. For example, allogeneic T-cell transplantation involves administering T cells obtained from a donor other than the subject to a subject.

[0084] “Cancer” refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. “Cancer” or “cancer tissue” can include tumors. Examples of cancers that can be treated by the methods of this disclosure include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, and other white blood cell malignancies. In some aspects, the methods of this disclosure can be used to reduce the size of tumors originating from, for example, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, malignant melanoma of the skin or eye, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease. Diseases, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) vegetations, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma. Sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), other B-cell malignancies, and combinations thereof. Certain cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory.

[0085] Refractory cancers are those that are not suitable for surgical intervention and that initially do not respond to chemotherapy or radiation therapy, or that become unresponsive over time.

[0086] As used in this article, "anti-tumor effect" refers to an increase in tumor volume, number of tumor cells, proliferation of tumor cells, number of metastases, overall survival or progression-free survival, life expectancy, or improvement in various tumor-related physiological symptoms. Anti-tumor effect can also refer to the prevention of tumor development, such as through vaccines.

[0087] As used herein, the term “progression-free survival” may be abbreviated as PFS, which refers to the time from the date of treatment to the date of disease progression or death from any cause according to the revised IWG criteria for response to malignant lymphoma.

[0088] As used in this article, “disease progression” or “progressive disease” may be abbreviated as PD, referring to the worsening of one or more symptoms associated with a specific disease. For example, disease progression in a subject with cancer may include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.

[0089] As used in this article, “duration of response” (DOR) refers to the time between a subject’s first objective response and the date of disease progression or death as confirmed by the revised IWG criteria for response to malignant lymphoma.

[0090] The term "overall survival" can be abbreviated as OS, and is defined as the time from the date of treatment to the date of death.

[0091] As used herein, a "cytokine" is a non-antibody protein released by a cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. Cytokines can be expressed endogenously by cells or administered to a subject. Immune cells (including macrophages, B cells, T cells, and mast cells) can release cytokines to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, while pro-inflammatory cytokines promote inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN)γ. Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-α, TNF-β, fibroblast growth factor (FGF) 2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute-phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0092] Chemokines are a type of cytokine that mediates cellular chemotaxis or directed movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eosinophil chemokine, eosinophil chemokine-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), γ-inducible protein 10 (IP-10), and thymus activation-regulated chemokines (TARC or CCL17).

[0093] Other examples of analytes and cytokines disclosed herein include, but are not limited to, chemokine (CC motif) ligands (CCL1), CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL13, IL-1, IL-3, IL-9, IL-11, IL-12, IL-14, IL-17, IL-20, IL-21, granulocyte colony-stimulating factor (G-CSF), leukemia suppressor factor (LIF), oncokinase M (OSM), CD154, lymphotoxin (LT) β, 4-1BB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-associated ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, and ligand 1 associated with TNF and ApoL leukocyte expression. (TALL-1) or TNF-associated apoptosis-inducing ligand (TRAIL).

[0094] The “therapeutic effective dose,” “effective dose,” “effective amount,” or “therapeutic effective dose” of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that has been demonstrated to protect a subject from disease onset or promote disease resolution by reducing the severity of disease symptoms, increasing the frequency and duration of asymptomatic periods of disease, or preventing injury or disability caused by the disease. The ability of a therapeutic agent to promote disease resolution can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0095] As used herein, the term “lymphocyte” includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic / cell toxic lymphocyte that represents a major component of the innate immune system. NK cells repel tumor cells and cells infected by viruses. They function through apoptosis, or programmed cell death. They are called “natural killers” because they can kill cells without activation. T cells play a major role in cell-mediated immunity (without antibody involvement). The T cell receptor (TCR) distinguishes T cells from other lymphocyte types. The thymus is a specialized organ of the immune system, primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells (i.e., stem memory T cells), and memory T cells. SCM Cells, like immature cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and exhibit many functional properties unique to memory cells; (ii) central memory T cells CM Cells express L-selectin and CCR7; they secrete IL-2 but not IFNγ or IL-4, and (iii) effector memory T cells. EM B cells, however, do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4, regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and γδ T cells. On the other hand, B cells play a major role in humoral immunity (with antibody involvement). B cells produce antibodies and antigens and act as antigen-presenting cells (APCs), transforming into memory B cells upon activation by antigen-antigen interactions. In mammals, immature B cells form in the bone marrow, from which their name originates.

[0096] The terms "genetically engineered" or "engineered" refer to methods of modifying the genome of cells, including but not limited to deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof. In some aspects, the modified cells are lymphocytes, such as CD3-expressing T cells, or modified cells that can be obtained from a patient or donor. Cells can be modified to express exogenous constructs, such as the T-cell receptor (TCR) disclosed herein, which are incorporated into the cell genome. In some aspects, cells are modified to express CD3.

[0097] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including alpha-1, cytokines, and complement) produced by any of these cells or the liver, which cause selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infecting cells or tissues, cancer cells or other abnormal cells, or (in the case of autoimmune or pathological inflammation) normal human cells or tissues in vertebrates.

[0098] The term "immunotherapy" refers to the treatment of a subject who has a disease or is at risk of contracting or relapsing into a disease by means of methods including inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T-cell therapy. T-cell therapy may include adoptive T-cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T-cell transplantation.

[0099] The cells used in the immunotherapy described herein can be derived from any source known in the art. For example, T cells can be differentiated from hematopoietic stem cell populations in vitro, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art (such as FICOLL™ isolation and / or apheresis). Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety. Immunotherapy may also include administering modified cells to a subject, wherein said modified cells express the CD3 and TCR disclosed herein. In some aspects, the modified cells are not T cells.

[0100] As used herein, “patient” includes anyone with cancer (e.g., lymphoma or leukemia). The terms “subject” and “patient” are used interchangeably in this document.

[0101] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can form the sequence of a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers both to short chains, such as those commonly referred to in the art as peptides, oligopeptides, and oligomers, and to longer chains, which are commonly referred to in the art as proteins, among many types. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0102] As used herein, “stimulus” refers to a primary response induced by the binding of a stimulating molecule to its homologous ligand, wherein this binding mediates a signal transduction event. A “stimulating molecule” is a molecule on T cells, such as the T cell receptor (TCR) / CD3 complex, which specifically binds to a homologous stimulating ligand present on antigen-presenting cells. A “stimulating ligand” is a stimulating molecule on T cells that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), can specifically bind to the stimulating molecule on T cells, thereby mediating primary T cell responses, including but not limited to activation, initiation of an immune response, and proliferation. Stimulating ligands include, but are not limited to, peptide-loaded MHC class I molecules, anti-CD3 antibodies, hyperagonist anti-CD2 antibodies, hyperagonist anti-CD28 antibodies, and hyperagonist anti-CD3 antibodies.

[0103] The terms “conditioning” and “preconditioning” are used interchangeably in this document and refer to preparing a patient who requires T-cell therapy for the appropriate condition. Conditioning, as used herein, includes, but is not limited to, reducing the number of endogenous lymphocytes prior to T-cell therapy, removing cytokine deposition, increasing serum levels of one or more homeostatic cytokines or pro-inflammatory factors, enhancing the effector function of T cells administered after conditioning, enhancing antigen-presenting cell activation and / or availability, or any combination thereof. In one aspect, "opsonization" includes increasing serum levels of one or more cytokines, such as interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), interleukin-10 (IL-10), interleukin-5 (IL-5), gamma-inducible protein-10 (IP-10), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), placental growth factor (PLGF), C-reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), or any combination thereof. In another aspect, "opsonization" includes increasing serum levels of IL-7, IL-15, IL-21, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

[0104] "Treatment" in a subject refers to any type of intervention or treatment administered to the subject, or the administration of an active agent, with the aim of reversing, alleviating, improving, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or symptom-related biochemical markers. In one aspect, "treatment" includes partial remission. In another aspect, "treatment" includes complete remission.

[0105] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a / an" should be understood to mean "one or more / a" of any of the described or enumerated components.

[0106] The terms “about” or “substantially comprise” refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by one of ordinary skill in the art, depending in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “substantially comprise” may mean within one or more standard deviations according to practice in the art. Alternatively, “about” or “substantially comprise” may refer to a range up to 10% (i.e., ±10%). For example, about 3 mg may include any amount between 2.7 mg and 3.3 mg (10%). Furthermore, particularly with respect to biological systems or processes, these terms may refer to values ​​up to an order of magnitude or up to five times. When a particular value or composition is provided in this application and claims, unless otherwise stated, it should be assumed that the meaning of “about” or “substantially comprise” is within an acceptable margin of error for that particular value or composition.

[0107] As stated herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer within the listed range and (where appropriate) its fractional value (such as one-tenth and one-hundredth of an integer).

[0108] The various aspects of this disclosure are further described in detail in the following sections.

[0109] II. Compositions disclosed herein This disclosure relates to a T-cell receptor (TCR) or its antigen-binding portion thereof that specifically binds to an epitope on HPV16 E6, a nucleic acid molecule encoding the TCR or its antigen-binding portion thereof, and a cell containing the TCR or the nucleic acid molecule. Some aspects of this disclosure relate to a method of treating cancer in a subject of need, comprising administering the TCR described herein to the subject. Some aspects of this disclosure relate to a method of treating cancer in a subject of need, comprising administering a nucleic acid encoding the TCR described herein to the subject. Some aspects of this disclosure relate to a method of treating cancer in a subject of need, comprising administering cells containing the TCR described herein to the subject. Other aspects of this disclosure relate to an HPV16 E6 epitope that the TCR binds to and an HLA class I molecule complexed with a peptide containing the HPV16 E6 epitope.

[0110] T-cell receptors, or TCRs, are molecules present on the surface of T cells or T lymphocytes that are responsible for recognizing fragments of antigens as peptides that bind to the major histocompatibility complex (MHC) molecule. The binding between TCRs and antigenic peptides has a relatively low affinity and is degenerate: that is, many TCRs recognize the same antigenic peptides, and many antigenic peptides are recognized by the same TCRs.

[0111] The TCR consists of two distinct protein chains (i.e., it is a heterodimer). In 95% of human T cells, the TCR is composed of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively), while in 5% of human T cells, the TCR is composed of gamma and delta (γ / δ) chains (encoded by TRG and TRD, respectively). This ratio varies during individual development and in disease states such as leukemia. The ratio also differs between species. Orthologs of four loci have been mapped across various species. Each locus can produce multiple polypeptides with constant and variable regions.

[0112] When the TCR binds to the antigenic peptide and MHC (peptide / MHC), T lymphocytes are activated through signal transduction (i.e., a series of biochemical events mediated by related enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors).

[0113] II.A. Nucleic acid molecules Certain aspects of this disclosure relate to nucleic acid molecules comprising (i) a first nucleotide sequence encoding a recombinant TCR or its antigen-binding portion (“anti-HPV16 E6 TCR”) that specifically binds to human HPV16 E6; and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR. In some aspects, the second nucleotide sequence is a non-naturally occurring sequence. In other aspects, the second nucleotide sequence is synthetic. In still other aspects, the second nucleotide sequence comprises a sequence of nucleotides targeting and encoding an endogenous TCR. In some aspects, the anti-HPV16 E6 TCR cross-competes with a reference TCR for binding to human HPV16 E6. In some aspects, the anti-HPV16 E6 TCR binds to the same human HPV16 E6 epitope or overlapping epitope as the reference TCR.

[0114] In some aspects, the reference TCR comprises an α chain and a β chain, wherein the α chain, β chain, or both thereof comprise one or more sequences presented in Tables 3A-3E. In some aspects, the reference TCR comprises an α chain and a β chain; wherein the α chain comprises complementarity-determining regions 1 (CDR1), CDR2, and CDR3; wherein the β chain comprises CDR1, CDR2, and CDR3; and wherein the reference TCR comprises the α chain CDR3 shown in SEQ ID NO: 7 and the β chain CDR3 shown in SEQ ID NO: 10. In some aspects, the α chain CDR1, CDR2, and CDR3 sequences are present in the amino acid sequence shown in SEQ ID NO: 1, and the reference TCR comprises the β chain CDR1, CDR2, and CDR3 sequences present in the amino acid sequence shown in SEQ ID NO: 2. In some aspects, the reference TCR comprises an α chain and a β chain, wherein the α chain comprises the amino acid sequence shown in SEQ ID NO: 1, and the β chain comprises the amino acid sequence shown in SEQ ID NO: 2.

[0115] Table 3A. TCR sequences of α and β chains - pairs 6 Table 3B. TCR α and β chains from C*07:02 / HPV-16 E6-specific TIL clones Table 3C. TCR α and β chains for 2 Table 3D. C*07 / HPV16-E6 specific TNFα + The TCR α chain sequence of T cell clones. Table 3. E. C*07 / HPV16-E6 specific TNFα + The TCR β chain sequence of T cell clones. In some aspects, the reference TCR comprises an α chain and a β chain; wherein the α chain comprises complementarity-determining regions 1 (CDR1), CDR2, and CDR3; wherein the β chain comprises CDR1, CDR2, and CDR3; and wherein the reference TCR comprises the α chain CDR3 shown in SEQ ID NO: 164 and the β chain CDR3 shown in SEQ ID NO: 10. In some aspects, the α chain CDR1, CDR2, and CDR3 sequences are present in the amino acid sequence shown in SEQ ID NO: 150, and the reference TCR comprises the β chain CDR1, CDR2, and CDR3 sequences present in the amino acid sequence shown in SEQ ID NO: 160. In some aspects, the reference TCR comprises an α chain and a β chain, wherein the α chain comprises the amino acid sequence shown in SEQ ID NO: 150, and the β chain comprises the amino acid sequence shown in SEQ ID NO: 160.

[0116] II.A.1. TCR encoded by the first nucleotide sequence This disclosure relates to a TCR encoded by a first nucleotide sequence described herein. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α chain and a β chain, wherein the α chain comprises variable regions containing α chain CDR1, α chain CDR2, and α chain CDR3; and wherein the β chain comprises variable regions containing β chain CDR1, β chain CDR2, and β chain CDR3. In some aspects, the anti-HPV16 E6 TCR comprises an α chain CDR3 containing the amino acid sequence (CAGQLYNQGGKLIF) shown in SEQ ID NO: 7. In some aspects, the anti-HPV16 E6 TCR comprises a β chain CDR3 containing the amino acid sequence (CASSLPLDGRLTYEQYF) shown in SEQ ID NO: 10. In some aspects, the non-CDR regions in the α chain and / or β chain are further modified, for example, by substitution or mutation of one, two, three, four, five, or six amino acids, such that the α chain and / or β chain are not naturally occurring. In some respects, substitution or mutation can improve the TCR described herein in a variety of ways (e.g., binding affinity, binding specificity, stability, viscosity, or any combination thereof).

[0117] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain CDR1, wherein the α-chain CDR1 of the anti-HPV16 E6 TCR comprises the amino acid sequence (SIFNT) as shown in SEQ ID NO: 5. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β-chain CDR1, wherein the β-chain CDR1 of the anti-HPV16 E6 TCR comprises the amino acid sequence (MDHEN) as shown in SEQ ID NO: 8.

[0118] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain CDR2, wherein the α-chain CDR2 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO: 6 (LYKAGEL). In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β-chain CDR2, wherein the β-chain CDR2 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO: 9 (SYDVKM).

[0119] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes an α-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the variable region of the α-chain amino acid sequence shown in SEQ ID NO: 1. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes an α-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the variable region of the α-chain amino acid sequence shown in SEQ ID NO: 1, wherein the anti-HPV16 E6 TCR includes an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 7. In some respects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence contains the α-chain variable region present in the α-chain amino acid sequence shown in SEQ ID NO: 1.

[0120] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the variable region of the β-chain amino acid sequence shown in SEQ ID NO: 2. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the variable region of the β-chain amino acid sequence shown in SEQ ID NO: 2, wherein the anti-HPV16 E6 TCR includes a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some respects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence contains the β-chain variable region present in the β-chain amino acid sequence shown in SEQ ID NO: 2.

[0121] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide also includes an α-chain constant region, a β-chain constant region, or both an α-chain constant region and a β-chain constant region. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes an α-chain constant region that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region of the α-chain amino acid sequence shown in SEQ ID NO: 1. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes an α-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the α-chain amino acid sequence shown in SEQ ID NO: 1, wherein the anti-HPV16 E6 TCR includes an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes the α-chain constant region present in the α-chain amino acid sequence shown in SEQ ID NO: 1. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence also includes an α-constant region that is different from an endogenous (e.g., naturally occurring) constant region of the α-chain. In some respects, the α-chain constant region comprises an amino acid sequence containing at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the α-chain shown in SEQ ID NO:1.

[0122] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region of the β-chain amino acid sequence shown in SEQ ID NO: 2. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the β-chain amino acid sequence shown in SEQ ID NO: 2, wherein the anti-HPV16 E6 TCR includes a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain constant region present in the amino acid sequence shown in SEQ ID NO: 2. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide also includes a β-constant region that is an endogenous (e.g., naturally occurring) constant region different from the β-chain. In some aspects, the β-chain constant region includes an amino acid sequence comprising at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the β-chain amino acid sequence shown in SEQ ID NO: 2.

[0123] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the α-chain amino acid sequence shown in SEQ ID NO: 1. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the α-chain amino acid sequence shown in SEQ ID NO: 1, wherein the anti-HPV16 E6 TCR comprises an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain containing the amino acid sequence shown in SEQ ID NO: 1.

[0124] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β-chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the β-chain amino acid sequence shown in SEQ ID NO: 2. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β-chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the β-chain amino acid sequence shown in SEQ ID NO: 2, wherein the anti-HPV16 E6 TCR comprises a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β-chain containing the amino acid sequence shown in SEQ ID NO: 2.

[0125] In some aspects, the anti-HPV16 E6 TCR comprises an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 164. In some aspects, the anti-HPV16 E6 TCR comprises a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the non-CDR regions in the α-chain and / or β-chain are further modified, for example, by substitution or mutation of one, two, three, four, five, or six amino acids, such that the α-chain and / or β-chain are not naturally occurring. In some aspects, substitution or mutation can improve the TCR described herein in various ways (e.g., binding affinity, binding specificity, stability, viscosity, or any combination thereof).

[0126] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain CDR1, wherein the α-chain CDR1 of the anti-HPV16 E6 TCR comprises an amino acid sequence as shown in SEQ ID NO: 162. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β-chain CDR1, wherein the β-chain CDR1 of the anti-HPV16 E6 TCR comprises an amino acid sequence as shown in SEQ ID NO: 8.

[0127] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain CDR2, wherein the α-chain CDR2 of the anti-HPV16 E6 TCR comprises an amino acid sequence as shown in SEQ ID NO: 163. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β-chain CDR2, wherein the β-chain CDR2 of the anti-HPV16 E6 TCR comprises an amino acid sequence as shown in SEQ ID NO: 9.

[0128] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes an α-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the variable region of the α-chain amino acid sequence shown in SEQ ID NO: 150. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes an α-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the variable region of the α-chain amino acid sequence shown in SEQ ID NO: 150, wherein the anti-HPV16 E6 TCR includes an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 164. In some respects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence contains the α-chain variable region present in the α-chain amino acid sequence shown in SEQ ID NO: 150.

[0129] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the variable region of the β-chain amino acid sequence shown in SEQ ID NO: 160. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the variable region of the β-chain amino acid sequence shown in SEQ ID NO: 160, wherein the anti-HPV16 E6 TCR includes a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some respects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence contains the β-chain variable region present in the β-chain amino acid sequence shown in SEQ ID NO: 160.

[0130] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide also includes an α-chain constant region, a β-chain constant region, or both an α-chain constant region and a β-chain constant region. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes an α-chain constant region that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region of the α-chain amino acid sequence shown in SEQ ID NO: 150. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes an α-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the α-chain amino acid sequence shown in SEQ ID NO: 150, wherein the anti-HPV16 E6 TCR includes an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 164. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes an α-chain constant region present in the α-chain amino acid sequence shown in SEQ ID NO: 150. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence also includes an α-constant region that is different from an endogenous (e.g., naturally occurring) constant region of the α-chain. In some respects, the α-chain constant region comprises an amino acid sequence containing at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the α-chain shown in SEQ ID NO: 150.

[0131] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region of the β-chain amino acid sequence shown in SEQ ID NO: 160. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the β-chain amino acid sequence shown in SEQ ID NO: 160, wherein the anti-HPV16 E6 TCR includes a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence includes a β-chain constant region present in the β-chain amino acid sequence shown in SEQ ID NO: 160. In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide also includes a β-constant region that is different from an endogenous (e.g., naturally occurring) constant region of the β-chain. In some aspects, the β-chain constant region includes an amino acid sequence comprising at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the β-chain amino acid sequence shown in SEQ ID NO: 160.

[0132] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the α-chain amino acid sequence shown in SEQ ID NO: 150. In other aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the α-chain amino acid sequence shown in SEQ ID NO: 150, wherein the anti-HPV16 E6 TCR comprises an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 164. In some respects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain containing the amino acid sequence shown in SEQ ID NO: 150.

[0133] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β-chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the β-chain amino acid sequence shown in SEQ ID NO: 160. In other aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some respects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises a β chain containing the amino acid sequence shown in SEQ ID NO:160.

[0134] In some respects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence comprises an α-chain constant region, a β-chain constant region, or both thereof; and wherein the α-chain constant region, the β-chain constant region, or both thereof comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five substitutions within the target sequence relative to the corresponding amino acid sequence of the endogenous TCR.

[0135] In some aspects, the α-chain of the anti-HPV16 E6 TCR encoded by the first nucleotide sequence also includes a signal peptide. Any signal peptide can be used in the anti-CCDN1 TCR α-chain disclosed herein. In some aspects, the signal peptide is a naturally occurring TCR α-chain signal peptide. In some aspects, the signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 21. In some aspects, the signal peptide is a heterologous signal peptide, for example, a signal peptide derived from a protein other than the TCR α-chain. In some aspects, the signal peptide is a synthetic signal peptide. In some aspects, the signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 20 or 22. In some aspects, the α chain of the HPV16 E6 TCR encoded by the first nucleotide sequence does not contain a signal peptide.

[0136] In some respects, the signal peptide of the α chain is encoded by a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 23 or 24.

[0137] In some aspects, the β-chain of the anti-HPV16 E6 TCR encoded by the first nucleotide sequence also includes a signal peptide. Any signal peptide can be used in the anti-CCDN1 TCR β-chain disclosed herein. In some aspects, the signal peptide is a naturally occurring TCR β-chain signal peptide. In some aspects, the signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 22. In some aspects, the signal peptide is a heterologous signal peptide, for example, a signal peptide derived from a protein other than the TCR β-chain. In some aspects, the signal peptide is a synthetic signal peptide. In some aspects, the signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 20 or 21. In some aspects, the β chain of the HPV16 E6 TCR encoded by the first nucleotide sequence does not contain a signal peptide.

[0138] In some respects, the signal peptide of the β chain is encoded by a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 23 or 24.

[0139] In some respects, each of the α and β chains of the anti-HPV16 E6 TCR encoded by the first nucleotide sequence also contains a signal peptide. In some respects, the signal peptide of the α chain is the same as that of the β chain. In some respects, the signal peptide of the α chain is different from that of the β chain.

[0140] II.A.2. Epitope In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide sequence binds to the same epitope as the reference TCR. In some aspects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6 comprising the amino acid sequence (AFRDLCIVY) shown in SEQ ID NO: 104. In some aspects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6 consisting of the amino acid sequence shown in SEQ ID NO: 104. In some aspects, the epitope comprises amino acid residues 53-61 of HPV16 E6 (SEQ ID NO: 104), for example, "HPV16 E6". 53-61 In some respects, the epitope is formed by amino acid residues 53-61 of HPV16 E6 (SEQ ID NO: 104), for example, "HPV16 E6 53-61 "composition.

[0141] In some aspects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6 comprising the amino acid sequence (FAFRDLCIVYRD) shown in SEQ ID NO: 97. In some aspects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6 consisting of an amino acid sequence as shown in SEQ ID NO: 97. In some aspects, the epitope comprises amino acid residues 52-63 of HPV16 E6 (SEQ ID NO: 97), for example, "HPV16 E6". 52-63 In some respects, the epitope is formed by amino acid residues 52-63 of HPV16 E6 (97), for example, "HPV16 E6 52-63 "composition.

[0142] In some aspects, an epitope is part of a larger polypeptide (e.g., a peptide comprising an epitope sequence and (i) one or more additional amino acids at the N-terminus of the epitope sequence and / or (ii) one or more additional amino acids at the C-terminus of the epitope sequence). In some aspects, the length of the polypeptide comprising the epitope is at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, at least about 45 amino acids, or at least about 50 amino acids. In some respects, the length of the polypeptide containing the epitope is at least about 5 to at least about 10, at least about 5 to at least about 15, at least about 5 to at least about 20, at least about 10 to at least about 15, at least about 10 to at least about 20, at least about 10 to at least about 25, at least about 10 to at least about 30, at least about 10 to at least about 35, at least about 10 to at least about 40, at least about 10 to at least about 45, at least about 10 to at least about 50, at least about 15 to at least about 20, at least about 15 to at least about 25, at least about 15 to at least about 30, at least about 15 to at least about 35, at least about 15 to at least about 40, at least about 15 to at least about 45, or at least about 15 to at least about 50 amino acids.

[0143] In some aspects, the polypeptide containing an epitope comprises the epitope and at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, or at least about 15 additional amino acids at the N-terminus of the epitope. In some aspects, the polypeptide containing an epitope comprises the epitope and at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, or at least about 15 additional amino acids at the C-terminus of the epitope.

[0144] In some respects, epitopes are associated with HLA class I molecules. The human leukocyte antigen (HLA) system (the major histocompatibility complex [MHC] in humans) is an important component of the immune system and is controlled by genes located on chromosome 6. It encodes cell surface molecules on T cells that specifically present antigenic peptides to T cell receptors (TCRs). (See also Overview of the Immune System). Antigen-presenting MHC molecules are divided into two main classes: class I MHC molecules and class II MHC molecules.

[0145] Class I MHC molecules are present as transmembrane glycoproteins on the surface of all nucleated cells. A complete class I molecule consists of an α-heavy chain that binds to a β-2 microglobulin molecule. The heavy chain comprises two peptide-binding domains, an Ig-like domain, and a transmembrane region with a cytoplasmic tail. The heavy chain of class I molecules is encoded by genes at the HLA-A, HLA-B, and HLA-C loci. T cells expressing CD8 molecules respond to class I MHC molecules. These lymphocytes typically possess cytotoxic functions, requiring them to recognize any infected cell. Because every nucleated cell expresses class I MHC molecules, all infected cells can act as antigen-presenting cells for CD8 T cells (the non-polymorphic portion of the class I heavy chain that binds to CD8). Some class I MHC genes encode non-classical MHC molecules, such as HLA-G (which plays a role in protecting the fetus from maternal immune responses) and HLA-E (which presents peptides to certain receptors on natural killer (NK) cells).

[0146] In some respects, HLA class 1 molecules are selected from the HLA-A, HLA-B, and HLA-C alleles. In some respects, HLA class 1 molecules are selected from the HLA-E, HLA-F, and HLA-G alleles. In some respects, HLA class 1 molecules are HLA-A alleles. In some respects, HLA class 1 molecules are HLA-B alleles. In some respects, HLA class 1 molecules are HLA-C alleles.

[0147] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any known allele can be used in this disclosure. An updated list of HLA alleles is available at hla.alleles.org / (last accessed: July 17, 2023). In some aspects, the HLA class 1 molecule is the HLA-C allele selected from the HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*07, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18 alleles. In some aspects, the HLA-C allele is the HLA-C*07 allele. In some respects, the HLA-C allele is the HLA-C*07:01 allele. In other respects, the HLA-C allele is the HLA-C*07:02 allele.

[0148] In some aspects, the HLA class I molecule is an HLA-C allele selected from the group consisting of: HLA-C*07:01:01, HLA-C*07:01:02, HLA-C*07:01:04, HLA-C*07:01:05, HLA-C*07:01:06, HLA-C*07:01:07, HLA-C*07:01:08, HLA-C*07:01:09, HLA-C*07:01:10, HLA-C*07:01:11, HLA-C*07:01:12, HLA-C*07:01:13, HLA-C*07:01:14, HLA-C*07:01:15, HLA-C*07:01:16, HLA-C*07:01:17, HLA-C*07:01:18, HLA-C*07:01:19, HLA-C*07:01:20, HLA-C*07:01:21, HLA-C*07:01:22, HLA-C*07:01:23, HLA-C*07:01:24, HLA-C*07:01:25, HLA-C*07:01:26, HLA-C*07:01:27, HLA-C*07:01:28, HLA-C*07:01:29, HLA-C*07:01:30, HLA-C*07:于1:31, HLA-C*07:01:32, HLA-C*07:01:33, HLA-C*07:01:34, HLA-C*07:01:35, HLA-C*07:01:36, HLA-C*07:01:37, HLA-C*07:01:38, HLA-C*07:01:39, HLA-C*07:01:40, HLA-C*07:01:41, HLA-C*07:01:42, HLA-C*07:01:43, HLA-C*07:01:44, HLA-C*07:01:45, HLA-C*07:01:46, HLA-C*07:01:47, HLA-C*07:01:48, HLA-C*07:01:49, HLA-C*07:01:50, HLA-C*07:01:51, HLA-C*07:01:52, HLA-C*07:01:53, HLA-C*07:01:54, HLA-C*07:01:55, HLA-C*07:01:56, HLA-C*07:01:57, HLA-C*07:01:58, HLA-C*07:01:59, HLA-C*07:01:60, HLA-C*07:01:61, HLA-C*07:01:62, HLA-C*07:01:63, HLA-C*07:01:64, HLA-C*07:01:65,HLA-C*07:01:66、HLA-C*07:01:67、HLA-C*07:01:68、HLA-C*07:01:69、HLA-C*07:01:70、HLA-C*07:01:71、HLA-C*07:01 :72、HLA-C*07:01:73、HLA-C*07:01:74、HLA-C*07:01:75、HLA-C*07:01:76、HLA-C*07:01:77、HLA-C*07:01:78、HLA-C*07 :01:79、HLA-C*07:01:80、HLA-C*07:01:81、HLA-C*07:01:82、HLA-C*07:01:83、HLA-C*07:01:84、HLA-C*07:01:85、HLA-C *07:01:86、HLA-C*07:01:87、HLA-C*07:01:88、HLA-C*07:01:89、HLA-C*07:01:90、HLA-C*07:01:91、HLA-C*07:01:92、HL A-C*07:01:93、HLA-C*07:01:94、HLA-C*07:01:95、HLA-C*07:01:96、HLA-C*07:01:97、HLA-C*07:01:98、HLA-C*07:01:99 、HLA-C*07:01:100、HLA-C*07:01:101、HLA-C*07:01:102、HLA-C*07:01:103、HLA-C*07:01:104、HLA-C*07:01:105、HLA-C *07:01:106, HLA-C*07:01:107, HLA-C*07:01:108, HLA-C*07:01:109, HLA-C*07:01:110, HLA-C*07:01:111, HLA-C*07:01:112, HLA-C*07:01:113, HLA-C*07:01:114, HLA-C*07:01:115, HLA-C*07:01:116, HLA-C*07:01:117 and HLA-C*07:01:118.

[0149] In some aspects, the HLA class I molecule is an HLA-C allele selected from the group consisting of: HLA-C*07:02:01, HLA-C*07:02:01, HLA-C*07:02:02, HLA-C*07:02:03, HLA-C*07:02:04, HLA-C*07:02:05, HLA-C*07:02:06, HLA-C*07:02:07, HLA-C*07:02:08, HLA-C*07:02:09, HLA-C*07:02:10, HLA-C*07:02:11, HLA-C*07:02:12, HLA-C*07:02:13, HLA-C*07:02:14, HLA-C*07:02:15, HLA-C*07:02:16, HLA-C*07:02:17, HLA-C*07:02:18, HLA-C*07:02:19, HLA-C*07:02:20, HLA-C*07:02:21, HLA-C*07:02:22, HLA-C*07:02:23, HLA-C*07:02:24, HLA-C*07:02:25, HLA-C*07:02:26, HLA-C*07:02:27, HLA-C*07:02:28, HLA-C*07:02:29, HLA-C*07:02:30, HLA-C*07:02:31, HLA-C*07:02:32, HLA-C*07:02:33, HLA-C*07:02:34, HLA-C*07:02:35, HLA-C*07:02:36:01, HLA-C*07:02:36:02, HLA-C*07:02:37, HLA-C*07:02:38, HLA-C*07:02:39, HLA-C*07:02:40, HLA-C*07:02:41, HLA-C*07:02:42, HLA-C*07:02:43, HLA-C*07:02:44, HLA-C*07:02:45, HLA-C*07:02:46, HLA-C*07:02:47, HLA-C*07:02:48, HLA-C*07:02:49, HLA-C*07:02:50, HLA-C*07:02:51, HLA-C*07:02:52, HLA-C*07:02:53, HLA-C*07:02:54, HLA-C*HLA-C*07:02:63、HLA-C*07:02:64、HLA-C*07:02:65、HLA-C*07:02:66、HLA-C*07:02:67、HLA-C*07:02:68、HLA-C*07:02:69、HLA-C*07:02:70、HLA-C*07:02:71、HLA-C*07:02:72、HLA-C*07:02:73、HLA-C*07:02:74、HLA-C*07:02:75、HLA-C*07:02:76、HLA-C*07:02:77、HLA-C*07:02:78、HLA-C*07 :02:79、HLA-C*07:02:80、HLA-C*07:02:81、HLA-C*07:02:82、HLA-C*07:02:83、HLA-C*07:02:84、HLA-C*07:02:85、HLA-C*07:02:86、HLA-C*07:02:87、HLA-C*07:02:88、HLA-C*07:02:89、HLA-C*07:02:90、HLA-C*07:02:91、HLA-C*07:02:92、HLA-C*07:02:93、HLA-C*07:02:94、HLA-C*07:02:95、H LA-C*07:02:96、HLA-C*07:02:97、HLA-C*07:02:98、HLA-C*07:02:99、HLA-C*07:02:100、HLA-C*07:02:101、HLA-C*07:02:102、HLA-C*07:02:103、HLA-C*07:02:104:01、HLA-C*07:02:104:02、HLA-C*07:02:105、HLA-C*07:02:106、HLA-C*07:02:107、HLA-C*07:02:108、HLA-C*07:02:109、HLA-C*0 7:02:110, HLA-C*07:02:111, HLA-C*07:02:112, HLA-C*07:02:113, HLA-C*07:02:114, HLA-C*07:02:115, HLA-C*07:02:116, HLA-C*07:02:117, HLA-C*07:02:118, HLA-C*07:02:119, HLA-C*07:02:120, HLA-C*07:02:121, HLA-C*07:02:122, HLA-C*07:02:123, HLA-C*07:02:124, HLA-C*07:02:125,HLA-C*07:02:126, HLA-C*07:02:127, HLA-C*07:02:128, HLA-C*07:02:129, HLA-C*07:02:130, HLA- C*07:02:131, HLA-C*07:02:132, HLA-C*07:02:133, HLA-C*07:02:134, HLA-C*07:02:135, HLA-C*07 :02:136, HLA-C*07:02:137, HLA-C*07:02:138, HLA-C*07:02:139, HLA-C*07:02:140, HLA-C*07:02: 141. HLA-C*07:02:142, HLA-C*07:02:143, HLA-C*07:02:144, HLA-C*07:02:145 and HLA-C*07:02:146. ,

[0150] II.A.3 Second nucleotide sequence The second nucleotide sequence of the nucleic acid molecule disclosed herein can be any sequence or can encode any polypeptide capable of inhibiting the expression of endogenous TCR. In some aspects, the second nucleotide sequence is one or more siRNAs. In some aspects, one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding a constant region of the endogenous TCR. In some aspects, one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding a constant region of the wild-type human TCR. In some aspects, one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding a constant region of the α chain of the wild-type TCR. In some aspects, one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding a constant region of the β chain of the wild-type TCR. In some aspects, one or more siRNAs include (i) one or more siRNAs complementary to a target sequence within the nucleotide sequence encoding a constant region of the α chain of the wild-type TCR, and (ii) one or more siRNAs complementary to a target sequence within the nucleotide sequence encoding a constant region of the β chain of the wild-type TCR.

[0151] In some aspects, one or more siRNAs comprise nucleotide sequences selected from the group consisting of SEQ ID NO: 25-28 (Table 4). In some aspects, a second nucleotide sequence of a nucleic acid molecule encodes one or more siRNAs, wherein one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the α chain of a wild-type TCR, and wherein one or more siRNAs comprise nucleic acid sequences shown in SEQ ID NO: 25 and 26.

[0152] Table 4. siRNA sequences In some aspects, the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the β chain of a wild-type TCR, and wherein the one or more siRNAs comprise the nucleic acid sequences shown in SEQ ID NO: 27 and 28. In some aspects, the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs comprise (i) one or more siRNAs complementary to a target sequence within a nucleotide sequence encoding a constant region of the α chain of a wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences shown in SEQ ID NO: 25 and 26; and (ii) one or more siRNAs complementary to a target sequence within a nucleotide sequence encoding a constant region of the β chain of a wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences shown in SEQ ID NO: 27 and 28.

[0153] In some aspects, the second nucleotide sequence of the nucleic acid molecule comprises SEQ ID NO: 25-28. In some aspects, the second nucleotide sequence comprises SEQ ID NO: 25-28, wherein one or more of SEQ ID NO: 25-28 are separated by one or more nucleic acids that do not encode siRNA. In some aspects, one or more siRNAs are selected from the siRNAs disclosed in U.S. Patent Publication No. 2010 / 0273213A1, which is incorporated herein by reference in its entirety.

[0154] In some respects, the second nucleotide sequence of a nucleic acid molecule encodes a protein that can suppress the expression of an endogenous (e.g., wild-type) TCR. In other respects, the second nucleotide sequence encodes Cas9.

[0155] In some respects, the second nucleotide sequence of the nucleic acid molecule disclosed in this paper is codon-optimized.

[0156] II.A.3 Carrier Certain aspects of this disclosure relate to vectors comprising the nucleic acid molecules disclosed herein. In some aspects, the vector is a viral vector. In some aspects, the vector is a viral particle or a virus. In some aspects, the vector is a mammalian vector. In some aspects, the vector is a bacterial vector.

[0157] In some respects, the vector is a retroviral vector. In some respects, the vector is a gamma retrovirus. In some respects, the vector is selected from the group consisting of: adenovirus vectors, lentiviruses, Sendai viruses, baculovirus vectors, Epstein-Barr virus vectors, papillomavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, or adeno-associated virus (AAV) vectors. In certain respects, the vector is an AAV vector. In some respects, the vector is a lentivirus. In certain respects, the vector is an AAV vector. In some respects, the vector is a Sendai virus. In some respects, the vector is a hybrid vector. Examples of hybrid vectors that may be used in this disclosure can be found in Huang and Kamihira. Biotechnol. Adv. 31(2) Reference :208-23(2103) is incorporated into this paper in its entirety by way of citation.

[0158] II.B. Recombinant T-cell receptor (TCR) Certain aspects of this disclosure relate to a recombinant T-cell receptor (TCR) that specifically binds to human HPV16 E6 or its antigen-binding portion (“anti-HPV16 E6 TCR”). In some aspects, the anti-HPV16 E6 TCR is encoded by a nucleic acid molecule disclosed herein.

[0159] In some aspects, the anti-HPV16 E6 TCR cross-competes with the reference TCR for binding to human HPV16 E6. In some aspects, the anti-HPV16 E6 TCR binds to the same or overlapping epitopes of human HPV16 E6 as the reference TCR. In some aspects, the reference TCR comprises an α chain and a β chain, and the α chain of the reference TCR comprises the amino acid sequence shown in SEQ ID NO: 1. In some aspects, the β chain of the reference TCR comprises the amino acid sequence shown in SEQ ID NO: 2.

[0160] In some aspects, the anti-HPV16 E6 TCR comprises an α chain and a β chain, wherein the α chain contains a constant region, and wherein the β chain contains a constant region; wherein the constant region of the α chain contains an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the α chain containing the amino acid sequence shown in SEQ ID NO: 1. In some aspects, the anti-HPV16 E6 TCR comprises an α chain and a β chain, wherein the α chain contains a constant region, and wherein the β chain contains a constant region; wherein the constant region of the β chain contains an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the β chain containing the amino acid sequence shown in SEQ ID NO: 2.

[0161] In some aspects, the anti-HPV16 E6 TCR comprises an α chain and a β chain, wherein the α chain comprises a constant region, and wherein the β chain comprises a constant region; wherein (i) the α chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of the α chain comprising the amino acid sequence shown in SEQ ID NO: 1; and (ii) the β chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of the β chain comprising the amino acid sequence shown in SEQ ID NO: 2.

[0162] In some aspects, the α chain of the anti-HPV16 E6 TCR includes variable regions containing α chain CDR1, α chain CDR2, and α chain CDR3; and the β chain of the anti-HPV16 E6 TCR includes variable regions containing β chain CDR1, β chain CDR2, and β chain CDR3. In some aspects, the anti-HPV16 E6 TCR includes α chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the anti-HPV16 E6 TCR includes β chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10.

[0163] In some aspects, the α-chain CDR1 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 5. In some aspects, the β-chain CDR1 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 8.

[0164] In some aspects, the α-chain CDR2 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the β-chain CDR2 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO: 9.

[0165] In some aspects, the anti-HPV16 E6 TCR includes an α-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the variable region of the α-chain amino acid sequence shown in SEQ ID NO: 1. In some aspects, the anti-HPV16 E6 TCR includes an α-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the variable region of the α-chain amino acid sequence shown in SEQ ID NO: 1, wherein the anti-HPV16 E6 TCR includes an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the anti-HPV16 E6 TCR includes the α-chain variable region present in the α-chain amino acid sequence shown in SEQ ID NO: 1.

[0166] In some aspects, the anti-HPV16 E6 TCR includes a β-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the variable region of the β-chain amino acid sequence shown in SEQ ID NO: 2. In some aspects, the anti-HPV16 E6 TCR includes a β-chain variable region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the variable region of the β-chain amino acid sequence shown in SEQ ID NO: 2, wherein the anti-HPV16 E6 TCR includes a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the anti-HPV16 E6 TCR includes the β-chain variable region present in the β-chain amino acid sequence shown in SEQ ID NO: 2.

[0167] In some aspects, the anti-HPV16 E6 TCR encoded by the first nucleotide also includes an α-chain constant region, a β-chain constant region, or both an α-chain constant region and a β-chain constant region. In some aspects, the anti-HPV16 E6 TCR includes an α-chain constant region that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region of the α-chain amino acid sequence shown in SEQ ID NO: 1. In some aspects, the anti-HPV16 E6 TCR includes an α-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the α-chain amino acid sequence shown in SEQ ID NO: 1, wherein the anti-HPV16 E6 TCR includes an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the anti-HPV16 E6 TCR includes the α-chain constant region present in the α-chain amino acid sequence shown in SEQ ID NO: 1. In some aspects, the anti-HPV16 E6 encoded by the first nucleotide... The TCR also includes an α-constant region that is an endogenous (e.g., naturally occurring) constant region different from that of the α chain. In some aspects, the α-chain constant region includes an amino acid sequence containing at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the α-chain shown in SEQ ID NO: 1.

[0168] In some aspects, the anti-HPV16 E6 TCR includes a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region of the β-chain amino acid sequence shown in SEQ ID NO: 2. In some aspects, the anti-HPV16 E6 TCR includes a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the β-chain amino acid sequence shown in SEQ ID NO: 2, wherein the anti-HPV16 E6 TCR includes a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the anti-HPV16 E6 TCR includes the β-chain constant region present in the β-chain amino acid sequence shown in SEQ ID NO: 2. In some respects, the first nucleotide encodes anti-HPV16 E6 The TCR also includes a β-constant region that is an endogenous (e.g., naturally occurring) constant region different from that of the β chain. In some aspects, the β-chain constant region includes an amino acid sequence containing at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the β-chain shown in SEQ ID NO: 2.

[0169] In some aspects, the anti-HPV16 E6 TCR comprises an α-chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the α-chain amino acid sequence shown in SEQ ID NO: 1. In some aspects, the anti-HPV16 E6 TCR comprises an α-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the anti-HPV16 E6 TCR comprises an α-chain containing the amino acid sequence shown in SEQ ID NO: 1.

[0170] In some aspects, the anti-HPV16 E6 TCR comprises a β-chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the β-chain amino acid sequence shown in SEQ ID NO: 2. In some aspects, the anti-HPV16 E6 TCR comprises a β-chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the anti-HPV16 E6 TCR comprises a β-chain containing the amino acid sequence shown in SEQ ID NO: 2.

[0171] In some respects, the anti-HPV16 E6 TCR comprises an α-chain constant region, a β-chain constant region, or both thereof; and wherein the α-chain constant region, the β-chain constant region, or both thereof comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five substitutions within the target sequence relative to the corresponding amino acid sequence of the endogenous TCR.

[0172] II.B.2. Epitope In some aspects, the anti-HPV16 E6 TCR binds to the same epitope as the reference TCR. In some aspects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6 comprising the amino acid sequence shown in SEQ ID NO: 104. In some aspects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6 consisting of the amino acid sequence shown in SEQ ID NO: 104. In some aspects, the epitope comprises amino acid residues 53-61 of HPV16 E6 (SEQ ID NO: 104), for example, "HPV16 E6". 53-61 In some respects, the epitope is composed of HPV16 E6 (SEQ ID NO: 104) amino acid residues 53-61, for example, "HPV16 E6 53-61 "composition.

[0173] In some aspects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6 comprising the amino acid sequence (FAFRDLCIVYRD) shown in SEQ ID NO: 97. In some aspects, the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6 consisting of an amino acid sequence as shown in SEQ ID NO: 97. In some aspects, the epitope comprises amino acid residues 52-63 of HPV16 E6 (SEQ ID NO: 97), for example, "HPV16 E6". 52-63 In some respects, the epitope is formed by amino acid residues 52-63 of HPV16 E6 (SEQ ID NO: 97), for example, "HPV16 E6 52-63 "composition.

[0174] In some respects, epitopes are complexed with HLA class I molecules. In some respects, HLA class 1 molecules are selected from HLA-A, HLA-B, and HLA-C alleles. In some respects, HLA class 1 molecules are selected from HLA-E, HLA-F, and HLA-G alleles. In some respects, HLA class 1 molecules are HLA-A alleles. In some respects, HLA class 1 molecules are HLA-B alleles. In some respects, HLA class 1 molecules are HLA-C alleles.

[0175] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any known allele can be used in this disclosure. An updated list of HLA alleles is available at hla.alleles.org / (last accessed: February 27, 2019). In some aspects, HLA class 1 molecules are selected from HLA-A, HLA-B, and HLA-C alleles. In some aspects, HLA class 1 molecules are selected from HLA-E, HLA-F, and HLA-G alleles. In some aspects, HLA class 1 molecules are HLA-A alleles. In some aspects, HLA class 1 molecules are HLA-B alleles. In some aspects, HLA class 1 molecules are HLA-C alleles.

[0176] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any known allele can be used in this disclosure. An updated list of HLA alleles is available at hla.alleles.org / (last accessed: July 17, 2023). In some aspects, the HLA class 1 molecule is the HLA-C allele selected from the HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*07, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18 alleles. In some aspects, the HLA-C allele is the HLA-C*07 allele. In some respects, the HLA-C allele is the HLA-C*07:01 allele. In other respects, the HLA-C allele is the HLA-C*07:02 allele.

[0177] In some aspects, the HLA class I molecule is an HLA-C allele selected from the group consisting of: HLA-C*07:01:01, HLA-C*07:01:02, HLA-C*07:01:04, HLA-C*07:01:05, HLA-C*07:01:06, HLA-C*07:01:07, HLA-C*07:01:08, HLA-C*07:01:09, HLA-C*07:01:10, HLA-C*07:01:11, HLA-C*07:01:12, HLA-C*07:01:13, HLA-C*07:01:14, HLA-C*07:01:15, HLA-C*07:01:16, HLA-C*07:01:17, HLA-C*07:01:18, HLA-C*07:01:19, HLA-C*07:01:20, HLA-C*07:01:21, HLA-C*07:01:22, HLA-C*07:01:23, HLA-C*07:01:24, HLA-C*07:01:25, HLA-C*07:01:26, HLA-C*07:01:27, HLA-C*07:01:28, HLA-C*07:01:29, HLA-C*07:01:30, HLA-C*07:01:31, HLA-C*07:01:32, HLA-C*07:01:33, HLA-C*07:01:34, HLA-C*07:01:35, HLA-C*07:01:36, HLA-C*07:01:37, HLA-C*07:01:38, HLA-C*07:01:39, HLA-C*07:01:40, HLA-C*07:01:41, HLA-C*07:01:42, HLA-C*07:01:43, HLA-C*07:01:44, HLA-C*07:01:45, HLA-C*07:01:46, HLA-C*07:01:47, HLA-C*07:01:48, HLA-C*07:01:49, HLA-C*07:01:50, HLA-C*07:01:51, HLA-C*07:01:52, HLA-C*07:01:53, HLA-C*07:01:54, HLA-C*07:01:55, HLA-C*07:01:56, HLA-C*07:01:57, HLA-C*07:01:58, HLA-C*07:01:59, HLA-C*07:01:60, HLA-C*07:01:61, HLA-C*07:01:62, HLA-C*07:01:63, HLA-C*07:01:64, HLA-C*07:01:65,HLA-C*07:01:66、HLA-C*07:01:67、HLA-C*07:01:68、HLA-C*07:01:69、HLA-C*07:01:70、HLA-C*07:01:71、HLA-C*07:01 :72、HLA-C*07:01:73、HLA-C*07:01:74、HLA-C*07:01:75、HLA-C*07:01:76、HLA-C*07:01:77、HLA-C*07:01:78、HLA-C*07 :01:79、HLA-C*07:01:80、HLA-C*07:01:81、HLA-C*07:01:82、HLA-C*07:01:83、HLA-C*07:01:84、HLA-C*07:01:85、HLA-C *07:01:86、HLA-C*07:01:87、HLA-C*07:01:88、HLA-C*07:01:89、HLA-C*07:01:90、HLA-C*07:01:91、HLA-C*07:01:92、HL A-C*07:01:93、HLA-C*07:01:94、HLA-C*07:01:95、HLA-C*07:01:96、HLA-C*07:01:97、HLA-C*07:01:98、HLA-C*07:01:99 、HLA-C*07:01:100、HLA-C*07:01:101、HLA-C*07:01:102、HLA-C*07:01:103、HLA-C*07:01:104、HLA-C*07:01:105、HLA-C *07:01:106, HLA-C*07:01:107, HLA-C*07:01:108, HLA-C*07:01:109, HLA-C*07:01:110, HLA-C*07:01:111, HLA-C*07:01:112, HLA-C*07:01:113, HLA-C*07:01:114, HLA-C*07:01:115, HLA-C*07:01:116, HLA-C*07:01:117 and HLA-C*07:01:118.

[0178] In some aspects, the HLA class I molecule is an HLA-C allele selected from the group consisting of: HLA-C*07:02:01, HLA-C*07:02:01, HLA-C*07:02:02, HLA-C*07:02:03, HLA-C*07:02:04, HLA-C*07:02:05, HLA-C*07:02:06, HLA-C*07:02:07, HLA-C*07:02:08, HLA-C*07:02:09, HLA-C*07:02:10, HLA-C*07:02:11, HLA-C*07:02:12, HLA-C*07:02:13, HLA-C*07:02:14, HLA-C*07:02:15, HLA-C*07:02:16, HLA-C*07:02:17, HLA-C*07:02:18, HLA-C*...HLA-C*07:02:63、HLA-C*07:02:64、HLA-C*07:02:65、HLA-C*07:02:66、HLA-C*07:02:67、HLA-C*07:02:68、HLA-C*07:02:69、HLA-C*07:02:70、HLA-C*07:02:71、HLA-C*07:02:72、HLA-C*07:02:73、HLA-C*07:02:74、HLA-C*07:02:75、HLA-C*07:02:76、HLA-C*07:02:77、HLA-C*07:02:78、HLA-C*07 :02:79、HLA-C*07:02:80、HLA-C*07:02:81、HLA-C*07:02:82、HLA-C*07:02:83、HLA-C*07:02:84、HLA-C*07:02:85、HLA-C*07:02:86、HLA-C*07:02:87、HLA-C*07:02:88、HLA-C*07:02:89、HLA-C*07:02:90、HLA-C*07:02:91、HLA-C*07:02:92、HLA-C*07:02:93、HLA-C*07:02:94、HLA-C*07:02:95、H LA-C*07:02:96、HLA-C*07:02:97、HLA-C*07:02:98、HLA-C*07:02:99、HLA-C*07:02:100、HLA-C*07:02:101、HLA-C*07:02:102、HLA-C*07:02:103、HLA-C*07:02:104:01、HLA-C*07:02:104:02、HLA-C*07:02:105、HLA-C*07:02:106、HLA-C*07:02:107、HLA-C*07:02:108、HLA-C*07:02:109、HLA-C*0 7:02:110, HLA-C*07:02:111, HLA-C*07:02:112, HLA-C*07:02:113, HLA-C*07:02:114, HLA-C*07:02:115, HLA-C*07:02:116, HLA-C*07:02:117, HLA-C*07:02:118, HLA-C*07:02:119, HLA-C*07:02:120, HLA-C*07:02:121, HLA-C*07:02:122, HLA-C*07:02:123, HLA-C*07:02:124, HLA-C*07:02:125,HLA-C*07:02:126, HLA-C*07:02:127, HLA-C*07:02:128, HLA-C*07:02:129, HLA-C*07:02:130, HLA- C*07:02:131, HLA-C*07:02:132, HLA-C*07:02:133, HLA-C*07:02:134, HLA-C*07:02:135, HLA-C*07 :02:136, HLA-C*07:02:137, HLA-C*07:02:138, HLA-C*07:02:139, HLA-C*07:02:140, HLA-C*07:02: 141. HLA-C*07:02:142, HLA-C*07:02:143, HLA-C*07:02:144, HLA-C*07:02:145 and HLA-C*07:02:146. ,

[0179] II.B.3. Bispecific molecules Certain aspects of this disclosure relate to a bispecific molecule comprising a first antigen-binding molecule and a second antigen-binding molecule, wherein the first antigen-binding molecule comprises a TCR disclosed herein or its antigen-binding portion. In some aspects, the first antigen-binding molecule comprises a single-stranded variable fragment (“scFv”). The term “bispecific molecule” can include a molecule that is specific to two antigens, one specific having a TCR disclosed herein or an antigen-binding portion of a TCR disclosed herein, and the other specific targeting another antigen, or more than two antigens, for example, three antigens, four antigens, or more antigens. Thus, in some aspects, the term “bispecific molecule” includes a multispecific molecule.

[0180] In some respects, the second antigen-binding molecule specifically binds to a protein expressed on the surface of a T cell. Any protein expressed on the surface of a T cell can be targeted by the bispecific antibody disclosed herein. In some respects, the protein expressed on the surface of a T cell is not expressed by other cells. In some respects, the protein expressed on the surface of a T cell is expressed on the surface of one or more other human immune cells. In some respects, the protein expressed on the surface of a T cell is expressed on the surface of one or more other human immune cells, but not on the surface of human non-immune cells. In some respects, the second antigen-binding molecule specifically binds to a protein expressed on the surface of a T cell selected from CD3, CD4, CD2, CD5, CD6, CD8, CD11a (LFA-1α), CD43, CD45, and CD53. In some respects, the second antigen-binding molecule specifically binds to CD3. In some respects, the second antigen-binding molecule specifically binds to CD4. In some respects, the second antigen-binding molecule contains scFv.

[0181] In some respects, the first antigen-binding molecule and the second antigen-binding molecule are linked or associated via covalent bonds. In other respects, the first antigen-binding molecule and the second antigen-binding molecule are linked via peptide bonds.

[0182] II.C. Cells expressing TCR Certain aspects of this disclosure relate to cells comprising the nucleic acid molecules disclosed herein, the vectors disclosed herein, the recombinant TCRs disclosed herein, the bispecific molecules disclosed herein, or any combination thereof. Any cell may be used in this disclosure.

[0183] In some respects, cells express CD3. CD3 expression can be natural, for example, CD3 can be expressed by a nucleic acid sequence that is expressed endogenously within the cell. For example, T cells, monocytes, macrophages, dendritic cells, and natural killer (NK) cells naturally express CD3. Therefore, in some respects, the cell is a T cell, monocyte, macrophage, dendritic cell, or natural killer cell. In some respects, the cell is a T cell selected from natural killer T (NKT) cells and innate lymphocytes (ILCs). In some respects, the cell is a monocyte. In some respects, the cell is a macrophage. In some respects, the cell is a dendritic cell.

[0184] In some respects, the T cells are isolated from human subjects. In some respects, the human subject is the same subject who will ultimately receive T-cell therapy. In other respects, the subject is a donor subject, where the donor subject is not the same subject who will receive T-cell therapy.

[0185] In some respects, the cell is a cell that does not naturally express CD3, wherein the cell has been modified to express CD3. In some respects, the cell contains a transgene encoding CD3, wherein the transgene is expressed by the cell. In some respects, the cell contains a transgene encoding a protein that activates the expression of endogenous CD3 carried out by the cell. In some respects, the cell contains a transgene encoding a protein or siRNA that acts as an inhibitor of CD3 expression in the cell. In some respects, the transgene is incorporated into the cell's genome. In some respects, the transgene is not incorporated into the cell's genome.

[0186] In some respects, the cells modified to express CD3 are isolated from human subjects. In some respects, the human subject is the same subject who will ultimately receive the cell therapy. In other respects, the subject is a donor subject, wherein the donor subject is not the same subject who will receive the cell therapy.

[0187] II.D. HLA class I molecules Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptide comprises an amino acid sequence selected from SEQ ID NO: 90-116. Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptide comprises an amino acid sequence selected from SEQ ID NO: 100-104. Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptide is composed of an amino acid sequence selected from SEQ ID NO: 100-104. Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptide comprises the amino acid sequence shown in SEQ ID NO: 102. In some aspects, the peptide is composed of the amino acid sequence shown in SEQ ID NO: 102. Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptide comprises the amino acid sequence shown in SEQ ID NO: 103. In some aspects, the peptide is composed of the amino acid sequence shown in SEQ ID NO: 103. Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptide comprises the amino acid sequence shown in SEQ ID NO: 104. In some respects, the peptide consists of the amino acid sequence shown in SEQ ID NO: 104.

[0188] Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptides comprise amino acid sequences selected from SEQ ID NO: 95-99. Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptides consist of amino acid sequences selected from SEQ ID NO: 95-99. Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptides comprise the amino acid sequence shown in SEQ ID NO: 97. In some aspects, the peptides consist of the amino acid sequence shown in SEQ ID NO: 97.

[0189] Some aspects of this disclosure relate to HLA class I molecules complexed with peptides, wherein the peptides comprise amino acid sequences selected from SEQ ID NO: 105-109.

[0190] In newspaper,HLA I, see HLA-A or HLA-B in HLA-C In the same form as HLA-E, HLA-F, HLA-G 1She was also shown as HLA-C*07:01:01 and HLA-C*07:01:02.HL A-C*07:01:04、HLA-C*07:01:05、HLA-C*07:01:06、HLA-C*07:01:07、 HLA-C*07:01:08、HLA-C*07:01:09、HLA-C*07:01:10、HLA-C*07:01:1 1、HLA-C*07:01:12、HLA-C*07:01:13、HLA-C*07:01:14、HLA-C*07:01 :15、HLA-C*07:01:16、HLA-C*07:01:17、HLA-C*07:01:18、HLA-C*07 :01:19、HLA-C*07:01:20、HLA-C*07:01:21、HLA-C*07:01:22、HLA-C* 07:01:23、HLA-C*07:01:24、HLA-C*07:01:25、HLA-C*07:01:26、HLA- C*07:01:27、HLA-C*07:01:28、HLA-C*07:01:29、HLA-C*07:01:30、HL A-C*07:01:31、HLA-C*07:01:32、HLA-C*07:01:33、HLA-C*07:01:34 、HLA-C*07:01:35、HLA-C*07:01:36、HLA-C*07:01:37、HLA-C*07:01: 38、HLA-C*07:01:39、HLA-C*07:01:40、HLA-C*07:01:41、HLA-C*07:0 1:42、HLA-C*07:01:43、HLA-C*07:01:44、HLA-C*07:01:45、HLA-C*07 :01:46、HLA-C*07:01:47、HLA-C*07:01:48、HLA-C*07:01:49、HLA-C* 07:01:50、HLA-C*07:01:51、HLA-C*07:01:52、HLA-C*07:01:53、HLA- C*07:01:54、HLA-C*07:01:55、HLA-C*07:01:56、HLA-C*07:01:57、HL A-C*07:01:58、HLA-C*07:01:59、HLA-C*07:01:60、HLA-C*07:01:61、HLA-C*07:01:62、HLA-C*07:01:63、HLA-C*07:01:64、HLA-C*07:01:65、HLA-C*07:01:66、HLA-C*07:01:67、HLA-C*07:01:68、HLA- C*07:01:69、HLA-C*07:01:70、HLA-C*07:01:71、HLA-C*07:01:72、HLA-C*07:01:73、HLA-C*07:01:74、HLA-C*07:01:75、HLA-C*07 :01:76、HLA-C*07:01:77、HLA-C*07:01:78、HLA-C*07:01:79、HLA-C*07:01:80、HLA-C*07:01:81、HLA-C*07:01:82、HLA-C*07:01: 83、HLA-C*07:01:84、HLA-C*07:01:85、HLA-C*07:01:86、HLA-C*07:01:87、HLA-C*07:01:88、HLA-C*07:01:89、HLA-C*07:01:90、HL A-C*07:01:91、HLA-C*07:01:92、HLA-C*07:01:93、HLA-C*07:01:94、HLA-C*07:01:95、HLA-C*07:01:96、HLA-C*07:01:97、HLA-C* 07:01:98、HLA-C*07:01:99、HLA-C*07:01:100、HLA-C*07:01:101、HLA-C*07:01:102、HLA-C*07:01:103、HLA-C*07:01:104、HLA-C* 07:01:105, HLA-C*07:01:106, HLA-C*07:01:107, HLA-C*07:01:108, HLA-C*07:01:109, HLA-C*07:01:110, HLA-C*07:01:111, HLA-C*07:01:112, HLA-C*07:01:113, HLA-C*07:01:114, HLA-C*07:01:115, HLA-C*07:01:116, HLA-C*07:01:117 and HLA-C*07:01:118.

[0191] In some aspects, the HLA class I molecule is an HLA-C allele selected from the group consisting of: HLA-C*07:02:01, HLA-C*07:02:01, HLA-C*07:02:02, HLA-C*07:02:03, HLA-C*07:02:04, HLA-C*07:02:05, HLA-C*07:02:06, HLA-C*07:02:07, HLA-C*07:02:08, HLA-C*07:02:09, HLA-C*07:02:10, HLA-C*07:02:11, HLA-C*07:02:12, HLA-C*07:02:13, HLA-C*07:02:14, HLA-C*07:02:15, HLA-C*07:02:16, HLA-C*07:02:17, HLA-C*07:02:18, HLA-C*07:02:19, HLA-C*07:02:20, HLA-C*07:02:21, HLA-C*07:02:22, HLA-C*07:02:23, HLA-C*07:02:24, HLA-C*07:02:25, HLA-C*07:02:26, HLA-C*07:02:27, HLA-C*07:02:28, HLA-C*07:02:29, HLA-C*07:02:30, HLA-C*07:02:31, HLA-C*07:02:32, HLA-C*07:02:33, HLA-C*07:02:34, HLA-C*07:02:35, HLA-C*07:02:36:01, HLA-C*07:02:36:02, HLA-C*07:02:37, HLA-C*07:02:38, HLA-C*07:02:39, HLA-C*07:02:40, HLA-C*07:02:41, HLA-C*07:02:42, HLA-C*07:02:43, HLA-C*07:02:44, HLA-C*07:02:45, HLA-C*07:02:46, HLA-C*07:02:47, HLA-C*07:02:48, HLA-C*07:02:49, HLA-C*07:02:50, HLA-C*07:02:51, HLA-C*07:02:52, HLA-C*07:02:53, HLA-C*07:02:54, HLA-C*07:02:55, HLA-C*07:02:56, HLA-C*07:02:57, HLA-C*07:02:58, HLA-C*07:02:59, HLA-C*07:02:60, HLA-C*07:02:61, HLA-C*07:02:62HLA-C*07:02:63、HLA-C*07:02:64、HLA-C*07:02:65、HLA-C*07:02:66、HLA-C*07:02:67、HLA-C*07:02:68、HLA-C*07:02:69、HLA-C*07:02:70、HLA-C*07:02:71、HLA-C*07:02:72、HLA-C*07:02:73、HLA-C*07:02:74、HLA-C*07:02:75、HLA-C*07:02:76、HLA-C*07:02:77、HLA-C*07:02:78、HLA-C*07 :02:79、HLA-C*07:02:80、HLA-C*07:02:81、HLA-C*07:02:82、HLA-C*07:02:83、HLA-C*07:02:84、HLA-C*07:02:85、HLA-C*07:02:86、HLA-C*07:02:87、HLA-C*07:02:88、HLA-C*07:02:89、HLA-C*07:02:90、HLA-C*07:02:91、HLA-C*07:02:92、HLA-C*07:02:93、HLA-C*07:02:94、HLA-C*07:02:95、H LA-C*07:02:96、HLA-C*07:02:97、HLA-C*07:02:98、HLA-C*07:02:99、HLA-C*07:02:100、HLA-C*07:02:101、HLA-C*07:02:102、HLA-C*07:02:103、HLA-C*07:02:104:01、HLA-C*07:02:104:02、HLA-C*07:02:105、HLA-C*07:02:106、HLA-C*07:02:107、HLA-C*07:02:108、HLA-C*07:02:109、HLA-C*0 7:02:110, HLA-C*07:02:111, HLA-C*07:02:112, HLA-C*07:02:113, HLA-C*07:02:114, HLA-C*07:02:115, HLA-C*07:02:116, HLA-C*07:02:117, HLA-C*07:02:118, HLA-C*07:02:119, HLA-C*07:02:120, HLA-C*07:02:121, HLA-C*07:02:122, HLA-C*07:02:123, HLA-C*07:02:124, HLA-C*07:02:125,HLA-C*07:02:126, HLA-C*07:02:127, HLA-C*07:02:128, HLA-C*07:02:129, HLA-C*07:02:130, HLA- C*07:02:131, HLA-C*07:02:132, HLA-C*07:02:133, HLA-C*07:02:134, HLA-C*07:02:135, HLA-C*07 :02:136, HLA-C*07:02:137, HLA-C*07:02:138, HLA-C*07:02:139, HLA-C*07:02:140, HLA-C*07:02: 141. HLA-C*07:02:142, HLA-C*07:02:143, HLA-C*07:02:144, HLA-C*07:02:145 and HLA-C*07:02:146. ,

[0192] In some aspects, HLA class I molecules comprise an HLA α chain and β2m. In some aspects, the HLA α chain comprises an α1 domain, an α2 domain, and an α3 domain. In some aspects, β2m comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17. In some aspects, the sequence of the HLA α chain is selected from any HLA protein sequence available at hla.alleles.org / (last accessed: February 27, 2019).

[0193] In some respects, HLA class I molecules are monomers. In some respects, HLA class I molecules are dimers. In some respects, HLA class I molecules are polymers. In some respects, HLA class I molecules are trimers. In some respects, HLA class I molecules are tetramers. In some respects, HLA class I molecules are pentamers.

[0194] Certain aspects of this disclosure relate to antigen-presenting cells (APCs) that contain any of the HLA class I molecules disclosed herein. In some aspects, the APC expresses an HLA class I molecule on its surface. In some aspects, the APC contains more than one HLA class I molecule disclosed herein.

[0195] II.E. Vaccine Some aspects of this disclosure relate to a cancer vaccine comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 90-116. Some aspects of this disclosure relate to a cancer vaccine comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 100-104. Some aspects of this disclosure relate to a cancer vaccine comprising a peptide composed of an amino acid sequence selected from SEQ ID NO: 100-104. Some aspects of this disclosure relate to a cancer vaccine comprising a peptide containing the amino acid sequence shown in SEQ ID NO: 102. In some aspects, the cancer vaccine comprises a peptide composed of the amino acid sequence shown in SEQ ID NO: 102. Some aspects of this disclosure relate to a cancer vaccine comprising a peptide containing the amino acid sequence shown in SEQ ID NO: 103. In some aspects, the cancer vaccine comprises a peptide composed of the amino acid sequence shown in SEQ ID NO: 103. Some aspects of this disclosure relate to a cancer vaccine comprising a peptide containing the amino acid sequence shown in SEQ ID NO: 104. In some aspects, the cancer vaccine comprises a peptide composed of the amino acid sequence shown in SEQ ID NO: 104.

[0196] Some aspects of this disclosure relate to a cancer vaccine comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 95-99. Some aspects of this disclosure relate to a cancer vaccine comprising a peptide consisting of an amino acid sequence selected from SEQ ID NO: 95-99. Some aspects of this disclosure relate to a cancer vaccine comprising a peptide containing an amino acid sequence shown in SEQ ID NO: 97. In some aspects, the cancer vaccine comprises a peptide consisting of an amino acid sequence shown in SEQ ID NO: 97.

[0197] Some aspects of this disclosure relate to a cancer vaccine comprising a peptide containing an amino acid sequence selected from SEQ ID NO: 105-109.

[0198] In some respects, vaccines also contain one or more excipients. In some respects, vaccines also contain one or more additional peptides. In some respects, one or more additional peptides contain one or more additional epitopes.

[0199] II.F. Immunoconjugates Some aspects of this disclosure relate to an immunoconjugate comprising (i) the TCR disclosed herein and (ii) a drug. Some aspects of this disclosure relate to an immunoconjugate comprising (i) the bispecific molecule disclosed herein and (ii) a drug. In some aspects, the drug comprises a cytotoxic agent. Any cytotoxic agent may be used in the immunoconjugate disclosed herein. In some aspects, the drug comprises maytansine-like substances, scabramycin, pyrrolobenzodiazepine, galicariin, amatoxins, analogues thereof, or derivatives thereof. In some aspects, the immunoconjugate comprises (i) the TCR disclosed herein and (ii) maytansine-like substances. In some aspects, the immunoconjugate comprises (i) the TCR disclosed herein and (ii) a maytansine-like analogue. In some aspects, the immunoconjugate comprises (i) the TCR disclosed herein and (ii) ansamitocin. In some aspects, the immunoconjugate comprises (i) the TCR disclosed herein and (ii) mertansine (DM1). In some respects, the immunoconjugate comprises (i) the TCR disclosed herein and (ii) ravtansine (DM4).

[0200] III. The Method of This Disclosure Some aspects of this disclosure relate to methods for treating cancer in subjects of need. Other aspects of this disclosure relate to methods for engineering antigen-targeting cells. Still other aspects of this disclosure relate to methods for enriching target T cell populations obtained from human subjects.

[0201] III.A. Methods of Treating Cancer Certain aspects of this disclosure relate to methods for treating cancer in a subject of need, comprising administering to the subject a nucleic acid molecule disclosed herein, a recombinant TCR disclosed herein, a bispecific molecule disclosed herein, an epitope disclosed herein, or an HLA class I molecule disclosed herein, or a vector or cell comprising any of the foregoing. In some aspects, the method comprises administering to the subject a nucleic acid molecule disclosed herein. In some aspects, the method comprises administering to the subject a recombinant TCR disclosed herein. In some aspects, the method comprises administering to the subject a bispecific molecule disclosed herein. In some aspects, the method comprises administering to the subject a disseminated epitope (e.g., a peptide comprising an amino acid sequence selected from SEQ ID NO: 90-116). In some aspects, the method comprises administering to the subject a vaccine disclosed herein. In some aspects, the method comprises administering to the subject a HLA class I molecule disclosed herein. In some aspects, the method comprises administering to the subject a vector disclosed herein. In some aspects, the method comprises administering to the subject a cell disclosed herein.

[0202] In some respects, cancer is HPV-positive cancer. In some respects, cancer is HPV-positive head and neck cancer. In some respects, cancer is selected from cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer, cervical cancer, pharyngeal cancer, oropharyngeal cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, metastatic cancer, HPV-related malignancies, HPV-related cancers, HPV-related cervical cancer, HPV-related squamous cell carcinoma, HPV-related adenocarcinoma, HPV-positive oropharyngeal squamous cell carcinoma, HPV-related vaginal adenocarcinoma, HPV-related adenosquamous carcinoma, HPV-related cervical adenocarcinoma, HPV-related anal squamous cell carcinoma, HPV-related penile squamous cell carcinoma, HPV-related vulvar squamous cell carcinoma, HPV-positive rectal squamous cell carcinoma, or any combination thereof.

[0203] In some ways, cancer is recurrent. In some ways, cancer is refractory. In some ways, cancer is advanced. In some ways, cancer is metastatic.

[0204] In some respects, the methods disclosed herein treat cancer in subjects. In some respects, the methods disclosed herein reduce the severity of one or more symptoms of cancer. In some respects, the methods disclosed herein reduce the size or number of tumors derived from said cancer. In some respects, the methods disclosed herein increase the overall survival of subjects compared to subjects not provided with the methods disclosed herein. In some respects, the methods disclosed herein increase the progression-free survival of subjects compared to subjects not provided with the methods disclosed herein. In some respects, the methods disclosed herein result in a partial response in subjects. In some respects, the methods disclosed herein result in a complete response in subjects.

[0205] In some aspects, the methods disclosed herein include treating cancer in a subject of need, the method comprising administering to the subject cells described herein, wherein the cells comprise nucleic acid molecules disclosed herein, vectors disclosed herein, recombinant TCRs disclosed herein, and / or bispecific antibodies disclosed herein. In some aspects, the cells are T cells. In some aspects, the cells are cells modified to express CD3.

[0206] In some respects, cells (e.g., T cells) are obtained from the subject. In other respects, cells (e.g., T cells) are obtained from a donor other than the subject.

[0207] In some respects, the subject is preconditioned prior to cell administration. Preconditioning may include any substance that promotes T cell function and / or survival. In some respects, preconditioning includes administering chemotherapy, cytokines, proteins, small molecules, or any combination thereof to the subject. In some respects, preconditioning includes administering interleukins. In some respects, preconditioning includes administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some respects, preconditioning includes administering cyclophosphamide, fludarabine, or both thereof. In some respects, preconditioning includes administering vitamin C, AKT inhibitors, ATRA (retinoic acid), rapamycin, or any combination thereof.

[0208] III.B. Methods for engineering antigen-targeting cells Certain aspects of this disclosure relate to methods for engineering antigen-targeting cells. In some aspects, the antigen is the HPV16 E6 antigen. In some aspects, the method includes transducing cells using a nucleic acid molecule or a vector disclosed herein. The cells can be any cells described herein. In some aspects, the cells are T cells described herein. In some aspects, the cells are cells modified to express CD3, as described herein. In some aspects, the cells (e.g., T cells) are obtained from a subject requiring T-cell therapy. In some aspects, the cells are obtained from a donor other than a subject requiring T-cell therapy. In some aspects, the cells are T cells or natural killer cells.

[0209] III.C. Methods for Enriching Target T Cell Populations Certain aspects of this disclosure relate to methods for enriching a population of target T cells obtained from a human subject. In some aspects, the method includes contacting T cells with an HLA class I molecule disclosed herein. In some aspects, the method includes contacting T cells with an APC disclosed herein. In some aspects, after contact, the enriched population of T cells contains a higher number of HLA class I-binding T cells relative to the number of T cells capable of binding HLA class I molecules prior to contact.

[0210] In some aspects, the method includes contacting T cells in vitro with a peptide, wherein the peptide comprises the amino acid sequence shown in SEQ ID NO: 104. In some aspects, the method includes contacting T cells in vitro with a peptide, wherein the peptide consists of the amino acid sequence shown in SEQ ID NO: 102, SEQ ID NO: 103, or SEQ ID NO: 104. In some aspects, the method includes contacting T cells in vitro with a peptide, wherein the peptide comprises the amino acid sequence shown in SEQ ID NO: 97. In some aspects, the method includes contacting T cells in vitro with a peptide, wherein the peptide consists of the amino acid sequence shown in SEQ ID NO: 97. In some aspects, after contact, the enriched T cell population comprises a higher number of T cells capable of binding HLA class I molecules compared to the number of T cells capable of binding HLA class I molecules prior to contact.

[0211] Some aspects of this disclosure relate to a method for selecting T cells capable of targeting tumor cells. In some aspects, the method includes contacting an isolated population of T cells in vitro with a peptide, wherein the peptide consists of an amino acid sequence as shown in SEQ ID NO: 102, SEQ ID NO: 103, or SEQ ID NO: 104. In some aspects, the method includes contacting an isolated population of T cells in vitro with a peptide, wherein the peptide consists of an amino acid sequence as shown in SEQ ID NO: 97. In some aspects, the T cells are obtained from a human subject.

[0212] T cells obtained from human subjects can be any T cells disclosed herein. In some respects, T cells obtained from human subjects are tumor-infiltrating lymphocytes (TILs).

[0213] In some aspects, the method also includes administering enriched T cells to a human subject. In some aspects, the subject is pre-conditioned prior to receiving the T cells as described herein.

[0214] All aspects, facets, and options described in this article can be combined in any and all variations.

[0215] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference, as each individual publication, patent or patent application is specifically and individually indicated to the extent of its inclusion herein by reference.

[0216] This disclosure has been generally described, and further understanding can be obtained by referring to the embodiments provided herein. These embodiments are for illustrative purposes only and are not intended to be limiting.

[0217] Example Example 1 Tumor-infiltrating lymphocytes (TILs) were isolated from biopsy specimens of primary early-stage tumor nodules from patients with head and neck squamous cell carcinoma and expanded in vitro using a non-clinical rapid expansion protocol (see Jin, J. et al., Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes in Gas-permeable Flasks to Numbers Needed for Patient Treatment. J. Immunother. 35, 283–292 (2012), which is incorporated herein by reference in its entirety). The HLA-C*07:02-restricted clonal T-cell responses of these TILs to human papillomavirus (HPV) converting protein E6 (HPV-E6, SEQ ID NO: 16) were detected. Characterization of antigen-specific T-cell responses and validation of T-cell receptor (TCR) specificity were performed through functional and structural analyses, respectively.

[0218] Multimer-based discovery—where clonal T-cell specificity is identified by flow cytometry-based pHLA-multimer staining of T cells—requires precise peptide sequences known (or predicted) to bind to the HLA allele of interest. These methodologies are not suitable for the unbiased discovery of TCRs and their homologous pHLA epitopes. Therefore, we integrated functional analysis and multimer-based techniques to identify novel TCR specificity and clone the HPV-E6 / C*07:02 TCR (see Nagarsheth, NB et al. TCR-engineered T cells targeting E7 for patients with metastatic HPV-associated epithelial cancers. Nat Med 1–7 (2021) doi:10.1038 / s41591-020-01225-1, which is incorporated herein by reference in its entirety).

[0219] For functional analysis, T cells were stimulated using artificial antigen-presenting cells (aAPCs) expressing a single HLA-C*07:02 allele. These C*07:02 aAPCs were capable of processing longer peptides and presenting epitopes via class I HLA molecules, and were pulsed with pre-synthesized peptides. For the discovery of HPV-E6 reactive T cells, primary TILs were stimulated with C*07:02 aAPCs pulsed with a 20-meric peptide spanning the entire E6 protein (Table 5). Following stimulation, more than 10% of the polyclonal TIL population was identified as E6 antigen (Ag)-specific C*07:02-restricted T cells, as indicated by Ag-dependent TNF-α production. Figure 1A-1C ).

[0220] Table 5. Overlapping eicoseptides throughout the chemosynthesis of the HPV16 E6 protein, highlighting the C*07:02-restricted T-cell reactive peptide epitope. To further elucidate the E6 antigenic peptide epitopes and identify the amino acid (AA) sequence loaded onto HLA-C*07:02, additional functional analysis of IFNγ was performed. Again, primary TILs were stimulated using C*07:02 aAPC of the peptide pulse. For this purpose, E6 icoseptides with overlapping 15 amino acids were organized into peptide matrix libraries (Tables 6 and 7). The twenty-nine icoseptides of E6 were arranged in a 6 × 5 rectangular grid, where columns were designated A through F and rows were designated G through K. For example, peptide matrix library D consisted of equal volumes of icoseptides 4, 10, 16, 22, and 28, and peptide matrix library H consisted of equal volumes of icoseptides 7, 8, 9, 10, 11, and 12. As with any other E6 peptide, peptide 10 was present in both peptide matrix libraries. Using the 11 peptide matrix libraries of E6, the 29 individual icoseptides were scanned to detect peptide Ag-dependent IFNγ production. Common sequences in peptides 10 and 11 were identified. 51 DFAFRDLCIVYRDGN 65 (SEQ ID NO: 90) Figure 2 (Tables 6 and 7). After identifying the peptide sequence as crucial for T cell response, the truncated deletion peptide spanning amino acid residues 51-65 of E6 was evaluated in primary TILs (Table 6). Figure 3-4 The desired peptide epitopes presented against the background of C*07:02 molecules were determined by multimer staining structure analysis. 52 FAFRDLCIVYRD 63 (SEQ ID NO:97) is crucial for TCR binding (Table 6). Importantly, it contains core residues. 54 FRDLCIVY 61A series of truncated peptides of (SEQ ID NO: 109) are recognized by primary TILs and can produce functional responses in primary TILs.

[0221] Table 6. Multimer staining and HPV E6-deficient peptides produced by IL-2 in Jurkat TCR-T cells To identify the dominant C*07-restricted antigenic peptide 51 DFAFRDLCIVYRDGN 65 The most functional TCR clone of (SEQ ID NO: 90) was used to separate multimer-positive and TNFα-positive cells using fluorescence-activated cell sorting (FACS). + TILs were used to molecularly clone the TCR α and β genes (Tables 3A-3E). Multimer staining of TILs promoted the segregation of individual TCR α-β heterodimers (Table 3A). However, the segregated TNFα... + Molecular cloning of TIL TCRs identified a heterologous clonal T cell pool with four TCR α chains and two β chains (Tables 3B-3E). Importantly, single α-β heterodimers sorted from multimers (Table 3A) were also present in this heterologous clonal population (Table 3B).

[0222] In order to obtain TNFα + The most functionally relevant α-β pairs were identified from clones sorted by FACS, and heterodimer function was tested for each possible combination of α and β pairs (Table 3B). For this purpose, a human T cell line with a null T cell nucleoprotein nuclear factor (NFAT)-enhanced green fluorescent protein (EGFP) (NFAT-EGFP) reporter gene TCR was used. The superior α-β pairs were identified using EGFP reporter gene signaling and T cell immunophenotype. Figure 5A-5T (and 6A-6B). Although all TCR α-β pairs produced NFAT reporter gene activity, pairs 2 and 6 induced the largest EGFP signal and the highest level of CD69 expression (Table 3B, ). Figures 6A-6B ).

[0223] To further validate the specificity and function of the cloned TCRs, TCR-engineered T cells (TCR-T) from pair 6 (Tables 3A-3B) were selected for more complete functional and structural analysis. This specific α-β heterodimer was chosen for further characterization due to its unexpectedly dominant function. Importantly, C*07:02 / HPV-E6 Jurkat TCR-T cells presented with C*07:02... 52 FAFRDLCIVYRD 63(SEQ ID NO: 97) showed strong IL-2 production upon stimulation. Figure 4 Furthermore, for the deletion of peptides 16-27, IL-2 production was significantly reduced. Figure 4 (Table 6) This indicates that 53 AFRDLCIVY 61 (SEQ ID NO:104) represents the smallest functionally optimal peptide Ag of this TCR. To further verify this, we evaluated the IFNγ functional affinity of this smallest epitope by adding one C-terminal or one N-terminal amino acid residue to either side as shown below: deletion of peptide 10 52 FAFRDLCIVY 61 (SEQ ID NO: 99), No. 14 53 AFRDLCIVYR 62 (SEQ ID NO: 103) and No. 15 53 AFRDLCIVY 61 (SEQ ID NO: 104). Although all three peptides showed dose-dependent activation of C*07:02 / HPV-E6 primary TCR-T, peptide 15 was missing. 53 AFRDLCIVY 61 (SEQ ID NO: 104) induces larger maximum IFNγ production and exhibits the lowest EC 50 ( Figure 7 Furthermore, when loaded with the missing peptide 15, C*07:02 / HPV-E6 JurkatTCR-T showed strong multimeric staining ( Figures 8A-8D ).

[0224] The HLA-C*07:02 allele shares 99.45% similarity with HLA-C*07:01, differing by only 2 amino acids (Table 7). Figure 9 Therefore, we investigated the C*07:02 / HPV-E6 TCR recognition of E6 presented by HLA-C*07:01. 53 AFRDLCIVY 61 (SEQ ID NO: 104; lacking peptide 15) epitope ability. When primary C*07:02 / HPV-E6 TCR-T cells were stimulated with peptide pulses of C*07:01 aAPC, IFNγ production increased in a dose-dependent manner. Furthermore, the amount of IFNγ produced after stimulation with C*07:01 aAPC was comparable to that produced after stimulation with C*07:02 aAPC. Figure 10 This indicates that the TCR binds to two alleles to produce a functional response in T cells.

[0225] Table 7. Amino acid sequences of HLA C*07:01 and C*07:02. These results clearly demonstrate that C*07:02 / HPV-E6 53-61 TCR-transduced T cells recognize the HPV oncogene E6, generating potent T cell function. The use of these newly cloned tumor-responsive C*07:02-restricted E6 α and β TCR genes could increase the global reach of HPV-targeted TCR gene therapy beyond those patients with the HLA-A*02:01 allele. Furthermore, when compared to the A*02:01 / E7 TCR with reported clinical activity, C*07:02 / HPV-E6… 53-61 TCR-transduced T cells exhibited superior function, as evidenced by maximal INF-γ production. Figure 8E-8F Surprisingly, these data indicate that C*07:02 / HPV-E6 53-61 This not only increases patient eligibility but also provides superior efficacy of TCR drug products.

[0226] method Cells and samples Human peripheral blood samples were commercially available, and tumor-infiltrating lymphocytes were obtained from cancer patients. Artificial antigen-presenting cells (aAPCs) are a derivative of the erythroleukemia cell line K562, exhibiting defective HLA gene expression. Jurkat 76 is a T-cell leukemia cell line engineered to express CD8 and lack endogenous T-cell receptor expression.

[0227] peptides All peptides were commercially available, received in lyophilized form, and reconstituted in DMSO at 50 μg / ml. The purity of the overlapping eicoseptide was confirmed to be >80%, and the purity of the deletion peptides used in multimer staining was confirmed to be >95%.

[0228] pHLA multimer staining for flow cytometry Soluble HLA (sHLA) monomers undergo peptide exchange followed by dimerization, and are then used directly to stain antigen-specific T cells from various sources. In short, during in vitro peptide exchange, His-labeled sHLA monomers are incubated with a high excess of the peptide of interest, saturating the monomers with the exchanged peptide. Then, the sHLA monomers loaded with the novel peptide are dimerized using an anti-His PE-conjugated mAb. The PE-sHLA peptide-exchanged dimer is then used to stain antigen-specific T cells.

[0229] T cells were pretreated with the tyrosine kinase inhibitor dasatinib to improve HLA multimer staining intensity by lowering the TCR / pHLA interaction affinity threshold required for cell staining. T cells were then stained with fluorescently labeled dimers. Cells were subsequently treated with TruStain FcX (Biolegend) Fc receptor blocker and stained with αCD8 (clone B9.11) and either the immobilizable viability dye eF506 (ThermoFisher) or DAPI (Sigma). Negative controls included either unexchanged or unexchanged (DMSO) sHLA monomers.

[0230] Flow cytometry and cell sorting T cell surface receptors were stained with sHLA dimer (PE), αCD8, and αCD3. Dead cells were identified using a viability-fixing dye. Stained cells were analyzed using CytoFLEX (Beckman Coulter), and data were analyzed using FlowJo™ software. Fluorescence-activated cell sorting (FACS) was performed on FACS Aria II (BD Bioscience).

[0231] Intracellular cytokine staining of TNFα in primary tumor-infiltrating lymphocytes (TILs) Thaw the cryopreserved TIL and use 4 × 10 6 Cells / ml were incubated in cRPMI medium supplemented with DNase at a 1:1000 dilution for at least 2 hours. Cell count and viability were checked immediately after thawing and after incubation in a NucleoCounter. Cells were incubated in cRPMI medium at 1 × 10⁻⁶ cells / ml. 6 One TIL and 2 × 10 5Co-stimulation was established in 96-well U-shaped plates containing an artificial APC expressing a single HLA allele C*07:02 (aAPC). At the start of all stimulation, all co-stimulation wells received co-stimulation antibodies (anti-CD28 and anti-CD49d, 1 μg / mL each; BioLegend catalog numbers 302934 and 304340, respectively) and GolgiPlug containing brefeldin A (1:1000 dilution; BD Biosciences catalog number 555029). TIL-aAPC co-stimulation was incubated overnight (16 hours) at 37°C with 5% CO2 humidification. For peptide stimulation, peptides from the entire HPV16 E6 peptide library were added to individual wells to a final concentration of 1 μg / mL for each individual peptide. As a negative control, DMSO was added to the cells at the same concentration used for peptide stimulation. For the positive control, anti-CD3 (clone OKT3, BioLegend catalog number 317326) antibody was added at a final concentration of 1 μg / ml. The final co-stimulation volume in each well was 200 μL.

[0232] Antibodies used for surface staining include anti-human CD3 PE-Cy7 (clone UCHT1, Biolegend catalog number 300420), anti-human CD4 AF-700 (clone RPA-T4, Biolegend catalog number 300526), ​​and anti-human CD8 FITC (clone B9.11, Beckman Coulter catalog number B76279). Antibodies used for intracellular staining include anti-human TNF-α APC (clone Mab11, Biolegend catalog number 502912).

[0233] Gene T cells positive for C*07:02 / E6 pHLA multimer staining (Table 6) were subjected to fluorescence-activated cell sorting (FACS) using FACS Aria (BD Biosciences) and the TCR gene was cloned. Cells were centrifuged to precipitate and RNA was extracted (Qiagen). The extracted RNA was subjected to 5' RACE (rapid amplification of cDNA), in which mRNA was converted to cDNA using reverse transcriptase (RT), oligo-dT linker primers, and GSP (Takara).

[0234] Subsequently, first-round and nested PCR of α, β1, and β2 were performed. For first-round PCR, the reverse primer bound to the 3' UTR region, and for nested PCR, the reverse primer bound to the 3' end of the coding region of the TCR constant region (Table 8). The PCR products were then run on 1.2% agarose gels, and bands of the correct size were identified on a transilluminator. These bands were then extracted and purified. The purified PCR products were then cloned into the pCR2.1 TOPO vector (ThermoFisher), transformed into Stellar cells, and plated overnight on amp plates containing X-gal and IPTG. Colonies were selected for small-scale preparation, and DNA was digested with EcoRI and run on 1% agarose gels. Bands of the correct size were sent for DNA sequencing. The sequencing data were analyzed, and the variable strand and CDR were identified according to IMGT nomenclature. The cloned endogenous α and β TCR gene sequences were identified as SEQ ID NO: 1 and 2.

[0235] Table 8. 5'-RACE primers Then, functionally codon-optimized TCRs were generated by synthesizing constructs (SEQ ID NO: 29-32) that encode α-strand sequences linked to β-strand sequences with self-cleaving microRNA T2A sequences, both with and without CD8α. Briefly, the synthetic TCR gene was amplified by PCR using NEB Q5 HotStart high-fidelity DNA polymerase (NEB; Table 9). The PCR product was run on a 0.8% gel, the corresponding bands were excised, and purified (Macherey Nagel). The purified PCR product was cloned into a pMSGV1 or pLenti plasmid backbone using an in-fusion reaction setup (Takara) with an insert:vector ratio of 2:1. The cloned product was then transformed into chemically competent *E. coli* Stellar™ cells (Takara) and plated overnight in LB ampicillin. After overnight culture, colonies were selected for Midi preparation (Macherey Nagel) and the sequences were validated. The cloned pMSGV1 (or pLenti) construct was then used for vector generation to deliver the viral TCR gene.

[0236] Table 9. In-fusion cloning primers * 680 bp; 20-mer, acting on both B1 and B2 as well as complete mouse. ** 1500 bp; 20-mer ***The furin protease linker OPT, 20-mer, also acts on complete mice. Production and determination of gamma retroviral vector (RVV) infection titer RVV production was achieved using a ping-pong method with stable PG13-producing cell lines. In short, phoenix-Eco (PhECO) cells (ATCCs) were transfected with a retroviral plasmid (pMSGV1) containing the TCR α-β heterodimer gene (SEQ ID NO: 18 and 19). Transiently generated monotropic virus from PhECO cells was collected and used to transduce the PG13 packaging cell lines (ATCCs). The RVV-containing supernatant from these stable PG13-producing cells was then harvested and titrated for TCR gene delivery to recipient T cells.

[0237] The RVV infection titer was then determined to quantify the number of functional retroviral particles in the transduced PG13 supernatant. Briefly, 96-well flat-bottom tissue culture plates were coated overnight with 12 μg / ml RetroNectin (Takara). RetroNectin was removed, and the plates were blocked with PBS containing 2% bovine serum albumin (BSA). The frozen PG13 supernatant was thawed on ice to produce serial dilutions of the virus. 150 μL of the serially diluted virus was added to the blocked RetroNectin-coated wells and centrifuged at 2000×g for 2 hours at 32°C. Once centrifugation was complete, any remaining virus was aspirated, and 5e4 TCR-invalidated Jurkat-76 cells were added and cultured at 37°C, 5% CO2 for 72 hours. Jurkat-76 cells were then harvested and stained with αCD3-PE antibody (BD Pharmingen). Cells were fixed in 2% paraformaldehyde for 20 minutes and analyzed using a flow cytometer (CytoFLEX, Beckman Coulter). Fluorescence readings of CD3 expression were used as readings of TCR transduction efficiency, and these readings were used to calculate the number of infectious units in the viral batch.

[0238] Jukrat TCR-engineered T cells (Jurkat TCR-T) Transduced the TCR-ineffective Jurkat / 76-CD8 (J76-CD8) cell line to express C*07:02 / HPV-E6. (53-61) Specific TCR. In short, it will contain antibodies against C*07:02 / HPV-E6. (53-61)(SEQ ID NO: 13) RVV titrated with a specific TCRα-β heterodimer was used for gene delivery to J76-CD8. As described above, non-tissue culture plates were coated with RetroNectin (Takara) and RVV, with the TCR gene of interest binding to the plate. J76-CD8 was then added to the RVV-coated plate for transduction. Transduced cells were harvested after 24 hours and transferred to tissue culture flasks for amplification for 72 hours, followed by TCR-positive sorting. For FACS, transduced cells were stained with αCD3-PE antibody (BDPharmingen) and sorted using MoFlo Astrios (Beckman Coulter). Sorted J76-CD8-C*07:02 / HPV-E6 cells were then processed. (53-61) The purity of the specific TCR-T is >90%.

[0239] T cells engineered with Jurkat NFAT-EGFP reporter gene TCR Invalidate TCR Jurkat / 76-CD8 (J76-CD8) 7 The cell line was transduced to express enhanced green fluorescent protein (EGFP) under the promoter of activating T cell nuclear factor (NFAT) transcription factor (TF). Six copies of the NFAT transcription factor binding site (TFBS) were engineered into the 5' promoter of the EGFP gene. Therefore, NFAT activation in this reporter gene line leads to NFAT binding to the transgene, thereby inducing EGFP protein transcription and translation, allowing indirect monitoring of T cell activation via EGFP fluorescence signaling in this TCR-inactive Jurkat T cell line.

[0240] TCR α and β chains were electroporated into the NFAT-EGFP reporter gene (Table 10), and this transient TCR expression system was subsequently implemented in downstream functional analyses. Using a Nucleofector™ II / 2b device (Lonza), electrical pulses were applied to Jurkat cells resuspended in a dedicated buffer (Amaxa® Nucleofector® Kit V) containing plasmids carrying the TCR α and β genes. Newly generated Jurkat lines were incubated at 37°C for 2 days when performing peptide-HLA multimer staining (for known antigens) or using peptide libraries to establish cocultures of HLA-matched aAPCs. Cocultures were then incubated for 48 hours. EGFP signaling was then monitored as a surrogate marker to determine whether the Jurkat lines carried functional TCR α-β heterodimers.

[0241] Table 10. NFAT-GFP reporter gene sequence T cells engineered from primary TCRs (primary TCR-T cells) Transduced peripheral blood T cells from healthy donors to express C*07:02 / HPV-E6 (53-61) Specific TCR. First, peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by diluting blood samples with equilibration solution into 50 mL leucosep filter conical tubes containing Ficoll. After centrifugation, the leukocyte layer of whole PBMCs was collected. CD8 T cells (purity >95%) were isolated from the PBMCs and activated with TransAct for 2 days according to the manufacturer's instructions. The TransAct was washed off, and the T cells were infected with retroviruses on RetroNectin-coated tissue culture plates. The T cells were then expanded for 7 days after transduction (day 9) and cryopreserved for future experiments. Simultaneously, the transduction efficiency of T cells was analyzed by multimer staining and flow cytometry.

[0242] IL-2 analysis was performed using ELISA for peptide epitope validation. Artificial APCs (aAPCs) expressing a single HLA allele C*07:02 were used as antigen-presenting cells. This was in contrast to J76-CD8-C*07:02 / HPV-E6. (53-61) Before co-culturing TCR-T, C*07:02 aAPC was pulsed at 37°C for 6 hours with HPV E6-deficient peptide (Table 6) at a concentration of 10 μg / mL. In short, 1 × 10 5 10 TCR-T cells and 1 × 10 5 C*07:02 aAPC cells were co-cultured together at a 1:1 ratio in 96-well U-bottom tissue culture plates. For positive controls, 100 ng / mL anti-CD3 antibody (OKT3) or 10 ng / mL PMA and 150 ng / mL Ca2+ were administered. 2+ Ionocarriers were used to stimulate TCR-T cells. For the negative control, TCR-T cells were co-cultured with aAPCs expressing C*07:02 that were either unpulsed or pulsed with irrelevant peptides. After stimulation at 37°C for 40 to 44 hours, the plates were centrifuged at 300g for 5 minutes and the supernatant was collected. The amount of IL-2 in the culture supernatant was measured using the Human IL-2 ELISA MAX Deluxe Kit (Biolegend) according to the manufacturer's instructions. The optical density (OD) values ​​at 450 nm and 570 nm were determined using a CLARIOstar Plus plate reader (BMG Labtech). Correction was performed by subtracting the OD value at 570 nm from the OD value at 450 nm.

[0243] IFNγ ELISpot In summary, 96-well plates were coated with anti-human IFN-g capture antibody. After overnight coating, the plates were washed and T-cell:aAPC co-cultures (Table 5) were added in the presence of the antigenic peptide and / or the corresponding control medium. After overnight incubation (12–15 h at 37°C / 5% CO2), the plates were washed to remove cells from the wells. Anti-human IFN-g (Biotin) detection antibody was then added to the wells to detect the captured IFN-g cytokine. The plates were then washed, followed by the addition of streptavidin-horseradish peroxidase (SA-HRP). Free SA-HRP was washed away, and a signal was generated by HRP enzymatic precipitation with AEC (3-amino-9-ethyl-carbazole) solution. The resulting signal (red spots) indicated the presence of an IFN-g producing cell or spot-forming unit (SFU). The number of SFUs was then counted using an ImmunoSpot® CTL analyzer. Results can be expressed as spot formation units per million cells (SFU / 10⁶ cells).

[0244] IFNγ functional affinity - validation of peptide epitopes in primary TCR-T cells IFNγ ELISApot was performed using 96-well plates with co-culture settings for TCR-dependent T cell activation. After coating with anti-human IFNγ capture antibody overnight, the plates were washed and primary TCR-T:aAPC co-culture was initiated. Primary C*07:02 / HPV-E6 cells were co-cultured in the presence of C*07:02 aAPCs with dose-response curves for different antigen-deficient peptides (Table 6), #10 (FAFRDLCIVY; SEQ ID NO: 99), #14 (AFRDLCIVYR; SEQ ID NO: 103), and #15 (AFRDLCIVY; SEQ ID NO: 104) in the presence of pulsed C*07:02 aAPCs and / or corresponding control media. 53-61 TCR-T were co-cultured.

Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a recombinant T-cell receptor (TCR) or its antigen-binding moiety ("anti-HPV16 E6 TCR") that specifically binds to human papillomavirus (HPV) converting protein E6 (HPV16 E6), The anti-HPV16 E6 TCR cross-competes with a reference TCR for binding to human HPV16 E6, and the reference TCR comprises an α chain and a β chain, wherein the α chain comprises an amino acid sequence as shown in SEQ ID NO: 1, and the β chain comprises an amino acid sequence as shown in SEQ ID NO:

2.

2. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a recombinant T-cell receptor (TCR) that specifically binds to human HPV16 E6 or its antigen-binding moiety ("anti-HPV16 E6 TCR"). The anti-HPV16 E6 TCR binds to the same human HPV16 E6 epitope or overlapping epitope as the reference TCR, and the reference TCR comprises an α chain and a β chain, wherein the α chain comprises an amino acid sequence as shown in SEQ ID NO: 1, and the β chain comprises an amino acid sequence as shown in SEQ ID NO:

2.

3. The isolated nucleic acid molecule as described in claim 1 or 2, wherein the anti-HPV16 E6 TCR binds to an epitope of HPV16 E6, the epitope of HPV16 E6 consisting of an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

4. The isolated nucleic acid molecule as described in claim 2 or 3, wherein the epitope is complexed with an HLA class I molecule.

5. The isolated nucleic acid molecule as described in claim 4, wherein the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele.

6. The isolated nucleic acid molecule as described in claim 4, wherein the HLA class I molecule is the HLA-C*07 allele.

7. The isolated nucleic acid molecule according to any one of claims 4 to 6, wherein the HLA class I molecule is the HLA-C*07:01 allele or the HLA-C*07:02 allele.

8. The isolated nucleic acid molecule according to any one of claims 1 to 7, wherein the anti-HPV16 E6 TCR comprises an α chain and a β chain. The α chain comprises a variable region containing α chain CDR1, α chain CDR2, and α chain CDR3; and The β chain contains variable regions comprising β chain CDR1, β chain CDR2 and β chain CDR3; The α-chain CDR3 contains an amino acid sequence as shown in SEQ ID NO:

7.

9. The isolated nucleic acid molecule of claim 8, wherein the β-chain CDR3 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

10.

10. The isolated nucleic acid molecule according to any one of claims 1 to 7, wherein the anti-HPV16 E6 TCR comprises an α chain and a β chain, wherein the α chain comprises a variable region containing α chain CDR1, α chain CDR2 and α chain CDR3; and The β chain contains variable regions comprising β chain CDR1, β chain CDR2 and β chain CDR3; The β-chain CDR3 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO:

10.

11. The isolated nucleic acid molecule of claim 10, wherein the α-chain CDR3 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

7.

12. The isolated nucleic acid molecule according to any one of claims 8 to 11, wherein the α-chain CDR1 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

5.

13. The isolated nucleic acid molecule according to any one of claims 8 to 12, wherein the β-chain CDR1 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

8.

14. The isolated nucleic acid molecule according to any one of claims 8 to 13, wherein the α-chain CDR2 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

6.

15. The isolated nucleic acid molecule according to any one of claims 8 to 14, wherein the β-chain CDR2 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

9.

16. The isolated nucleic acid molecule according to any one of claims 8 to 15, wherein the α-chain variable region of the anti-HPV16 E6 TCR comprises the amino acid sequence of the variable region present in the amino acid sequence shown in SEQ ID NO:

1.

17. The isolated nucleic acid molecule according to any one of claims 8 to 16, wherein the β-chain variable region of the anti-HPV16 E6 TCR comprises the amino acid sequence of the variable region present in the amino acid sequence shown in SEQ ID NO:

2.

18. The isolated nucleic acid molecule of any one of claims 8 to 17, wherein the α-chain of the anti-HPV16 E6 TCR further comprises a constant region, wherein the constant region is different from the endogenous constant region of the α-chain.

19. The isolated nucleic acid molecule of any one of claims 8 to 18, wherein the α chain of the anti-HPV16 E6 TCR further comprises a constant region, wherein the constant region of the α chain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region present in the amino acid sequence shown in SEQ ID NO:

1.

20. The isolated nucleic acid molecule of claim 18 or 19, wherein the α-chain constant region comprises an amino acid sequence comprising at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO:

1.

21. The isolated nucleic acid molecule according to any one of claims 8 to 20, wherein the β chain of the anti-HPV16 E6 TCR further comprises a constant region, wherein the constant region is different from the endogenous constant region of the β chain.

22. The isolated nucleic acid molecule of any one of claims 8 to 21, wherein the β chain of the anti-HPV16 E6 TCR further comprises a constant region, wherein the β chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region present in the amino acid sequence shown in SEQ ID NO:

2.

23. The isolated nucleic acid molecule of claim 21 or 22, wherein the β-chain constant region comprises an amino acid sequence comprising at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO:

2.

24. The isolated nucleic acid molecule according to any one of claims 8 to 23, wherein the α chain of the anti-HPV16 E6 TCR comprises an amino acid sequence as shown in SEQ ID NO:

1.

25. The isolated nucleic acid molecule of any one of claims 8 to 24, wherein the β chain of the anti-HPV16 E6 TCR comprises an amino acid sequence as shown in SEQ ID NO:

2.

26. The isolated nucleic acid molecule according to any one of claims 1 to 25, further comprising a second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of endogenous TCR.

27. The isolated nucleic acid molecule of claim 26, wherein the second nucleotide sequence is one or more siRNAs that reduce the expression of endogenous TCR.

28. The isolated nucleic acid molecule of claim 27, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding the constant region of the endogenous TCR.

29. The isolated nucleic acid molecule of claim 27 or 28, wherein the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 53-56.

30. The isolated nucleic acid molecule according to any one of claims 1 to 29, wherein the anti-HPV16 E6 TCR comprises an α-chain constant region, a β-chain constant region, or both thereof; and wherein the α-chain constant region, the β-chain constant region, or both thereof comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five substituted amino acid sequences within the target sequence relative to the corresponding amino acid sequence of the endogenous TCR.

31. The isolated nucleic acid molecule according to any one of claims 1 to 30, wherein the α chain comprises a signal peptide, the β chain comprises a signal peptide, or both the α chain and the β chain comprise a single peptide.

32. The isolated nucleic acid molecule of claim 31, wherein the signal peptide comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 20-22 and any combination thereof.

33. A vector comprising a nucleic acid molecule as described in any one of claims 1 to 32.

34. The vector as described in claim 33, wherein it is a viral vector, a mammalian vector, or a bacterial vector.

35. The vector as described in claim 33 or 34, wherein it is a retroviral vector.

36. The vector according to any one of claims 33 to 35, wherein the vector is selected from the group consisting of: adenovirus vector, lentivirus, Sendai virus vector, baculovirus vector, Epstein-Barr virus vector, papillomavirus vector, vaccinia virus vector, herpes simplex virus vector, mixed vector or adeno-associated virus (AAV) vector.

37. The vector as described in any one of claims 33 to 36, wherein it is a lentivirus.

38. A T-cell receptor (TCR) or its antigen-binding portion, comprising the α-chain variable region of the anti-HPV16 E6 TCR as claimed in any one of claims 8 to 32 and the β-chain variable region of the anti-HPV16 E6 TCR as claimed in any one of claims 8 to 32.

39. A recombinant T-cell receptor (TCR) that specifically binds to human HPV16 E6 or its antigen-binding portion ("anti-HPV16E6 TCR"), which cross-competes with a reference TCR for binding to human HPV16 E6; The reference TCR comprises an α chain and a β chain, wherein the α chain comprises the amino acid sequence shown in SEQ ID NO: 1, and the β chain comprises the amino acid sequence shown in SEQ ID NO: 2; and The anti-HPV16 E6 TCR comprises an α chain and a β chain, wherein the α chain contains a constant region, and wherein the β chain contains a constant region; wherein (i) The α-chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO: 1, or (ii) The β-chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence of SEQ ID NO:

2.

40. A recombinant T-cell receptor (TCR) that specifically binds to human HPV16 E6 or its antigen-binding portion ("anti-HPV16E6 TCR"), which binds to the same human HPV16 E6 epitope or overlapping epitope as a reference TCR; The reference TCR comprises an α chain and a β chain, wherein the α chain comprises the amino acid sequence shown in SEQ ID NO: 1, and the β chain comprises the amino acid sequence shown in SEQ ID NO: 2; and The anti-HPV16 E6 TCR comprises an α chain and a β chain, wherein the α chain contains a constant region, and wherein the β chain contains a constant region; wherein (i) The α-chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO: 1, or (ii) The β-chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence shown in SEQ ID NO:

2.

41. The anti-HPV16 E6 TCR of claim 39 or 40, which binds to an epitope of HPV16 E6, said epitope being composed of an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

42. The anti-HPV16 E6 TCR as described in claim 40 or 41, wherein the epitope is complexed with an HLA class I molecule.

43. The anti-HPV16 E6 TCR as described in claim 42, wherein the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele.

44. The anti-HPV16 E6 TCR as described in claim 42 or 43, wherein the HLA class I molecule is the HLA-C*07 allele.

45. The anti-HPV16 E6 TCR as described in any one of claims 42 to 44, wherein the HLA class I molecule is the HLA-C*07:01 allele or the HLA-C*07:02 allele.

46. ​​The anti-HPV16 E6 TCR as described in any one of claims 39 to 45, wherein the α chain of the anti-HPV16 E6 TCR comprises a variable region containing α chain CDR1, α chain CDR2, and α chain CDR3; and The β chain of the anti-HPV16 E6 TCR contains a variable region comprising β chain CDR1, β chain CDR2 and β chain CDR3; The α-chain CDR3 of the anti-HPV16 E6 contains an amino acid sequence as shown in SEQ ID NO:

7.

47. The anti-HPV16 E6 TCR of claim 46, wherein the β-chain CDR3 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

10.

48. The anti-HPV16 E6 TCR of any one of claims 39 to 45, wherein the α chain of the anti-HPV16 E6 TCR comprises a variable region containing α chain CDR1, α chain CDR2 and α chain CDR3; The β chain of the anti-HPV16 E6 TCR contains variable regions comprising β chain CDR1, β chain CDR2, and β chain CDR3; and The β-chain CDR3 of the anti-HPV16 E6 TCR contains the amino acid sequence shown in SEQ ID NO:

10.

49. The anti-HPV16 E6 TCR of claim 48, wherein the α-chain CDR3 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

7.

50. The anti-HPV16 E6 TCR of claim 49, wherein the α-chain CDR1 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

5.

51. The anti-HPV16 E6 TCR of any one of claims 46 to 50, wherein the β-chain CDR1 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

8.

52. The anti-HPV16 E6 TCR of any one of claims 46 to 51, wherein the α-chain CDR2 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

6.

53. The anti-HPV16 E6 TCR of any one of claims 46 to 52, wherein the β-chain CDR2 of the anti-HPV16 E6 TCR comprises the amino acid sequence shown in SEQ ID NO:

9.

54. The anti-HPV16 E6 TCR according to any one of claims 46 to 53, wherein the α-chain variable region of the anti-HPV16 E6 TCR comprises the amino acid sequence of the variable region present in the amino acid sequence shown in SEQ ID NO:

1.

55. The anti-HPV16 E6 TCR according to any one of claims 46 to 54, wherein the β-chain variable region of the anti-HPV16 E6 TCR comprises the amino acid sequence of the variable region present in the amino acid sequence shown in SEQ ID NO:

2.

56. The anti-HPV16 E6 TCR of any one of claims 39 to 55, wherein the α-chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence present in the constant region shown in SEQ ID NO:

1.

57. The anti-HPV16 E6 TCR of any one of claims 39 to 56, wherein the β-chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence present in the constant region shown in SEQ ID NO:

2.

58. The anti-HPV16 E6 TCR of any one of claims 39 to 57, wherein the α chain of the anti-HPV16 E6 TCR comprises an amino acid sequence as shown in SEQ ID NO:

1.

59. The anti-HPV16 E6 TCR of any one of claims 39 to 58, wherein the β chain of the anti-HPV16 E6 TCR comprises an amino acid sequence as shown in SEQ ID NO:

2.

60. The anti-HPV16 E6 TCR of any one of claims 39 to 59, wherein the α chain comprises a signal peptide, the β chain comprises a signal peptide, or both the α chain and the β chain comprise a single peptide.

61. The anti-HPV16 E6 TCR of claim 60, wherein the signal peptide comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 20-22 and any combination thereof.

62. A bispecific molecule comprising a first antigen-binding molecule and a second antigen-binding molecule, wherein the first antigen-binding molecule comprises a TCR or its antigen-binding portion as described in any one of claims 38 to 61.

63. The bispecific molecule of claim 62, wherein the first antigen-binding molecule comprises a single-stranded variable fragment ("scFv").

64. The bispecific molecule of claim 62 or 63, wherein the second antigen-binding molecule specifically binds to a protein expressed on the surface of a T cell.

65. The bispecific molecule of any one of claims 62 to 64, wherein the second antigen-binding molecule specifically binds to CD3.

66. The bispecific molecule of any one of claims 62 to 65, wherein the second antigen-binding molecule comprises scFv.

67. The bispecific molecule of any one of claims 62 to 66, wherein the first antigen-binding molecule and the second antigen-binding molecule are linked or associated by a covalent bond.

68. The bispecific molecule of any one of claims 62 to 67, wherein the first antigen-binding molecule and the second antigen-binding molecule are linked by a peptide bond.

69. A cell comprising a nucleic acid molecule as claimed in any one of claims 1 to 32, a vector as claimed in any one of claims 33 to 37, a TCR as claimed in claim 38, a recombinant TCR as claimed in any one of claims 39 to 61, or a bispecific molecule as claimed in any one of claims 62 to 68.

70. The cell of claim 69, further expressing CD3.

71. The cell of claim 69 or 70, selected from the group consisting of: T cells, natural killer (NK) cells, natural killer T (NKT) cells, or ILC cells.

72. An immunoconjugate comprising (i) a TCR as described in any one of claims 38 to 61 or a bispecific molecule as described in any one of claims 62 to 68 and (ii) a drug.

73. The immunoconjugate of claim 72, wherein the drug comprises a cytotoxic agent.

74. The immunoconjugate of claim 72 or 73, wherein the drug comprises maytansine, salicylic acid, pyrrolobenzodiazepine, galicariin, amatoxins, or analogues thereof.

75. A method of treating cancer in a subject in need, comprising administering to the subject a nucleic acid as described in any one of claims 1 to 32, a vector as described in any one of claims 33 to 37, a TCR as described in any one of claims 38 to 61, a bispecific molecule as described in any one of claims 62 to 68, a cell as described in any one of claims 69 to 71, an immunoconjugate as described in any one of claims 72 to 74, or any combination thereof.

76. The method of claim 75, wherein the cancer is selected from the group consisting of: HPV-positive head and neck cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer, or any combination thereof.

77. The method of claim 75 or 76, wherein the cancer is recurrent or refractory.

78. The method of any one of claims 75 to 77, wherein the cancer is locally advanced.

79. The method of any one of claims 75 to 78, wherein the cancer is advanced.

80. The method of any one of claims 75 to 79, wherein the cancer is metastatic.

81. The method of any one of claims 75 to 80, wherein the cells are obtained from the subject.

82. The method of any one of claims 75 to 81, wherein the cells are obtained from a donor other than the subject.

83. The method of any one of claims 75 to 82, wherein the subject is pre-conditioned prior to administration of the cells.

84. The method of claim 83, wherein the preconditioning comprises administering chemotherapy, cytokines, proteins, small molecules, or any combination thereof to the subject.

85. The method of claim 83 or 84, wherein the preconditioning includes the administration of interleukin.

86. The method of any one of claims 83 to 85, wherein the preconditioning comprises administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21 or any combination thereof.

87. The method of any one of claims 83 to 86, wherein the preconditioning comprises administering a preconditioning agent selected from the group consisting of cyclophosphamide, fludarabine, vitamin C, AKT inhibitors, ATRA, rapamycin, or any combination thereof.

88. The method of any one of claims 83 to 87, wherein the preconditioning comprises the application of cyclophosphamide, fludarabine, or both.

89. A method for engineering antigen-targeting cells, comprising transducing cells collected from a subject requiring T-cell therapy with a nucleic acid molecule as described in any one of claims 1 to 32 or a vector as described in any one of claims 33 to 37.

90. The method of claim 89, wherein the antigen-targeting cell further expresses CD3.

91. The method of claim 89 or 90, wherein the cell is a T cell or a natural killer (NK) cell.

92. An HLA class I molecule complexed with a peptide, wherein the HLA class I molecule comprises an α chain and a β chain; and wherein the peptide consists of an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

93. The HLA class I molecule as described in claim 92, wherein it is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele.

94. The HLA class I molecule as described in claim 92 or 93, wherein it is an HLA-C allele.

95. The HLA class I molecule as described in claim 92 or 93, wherein it is the HLA-C*07 allele.

96. The HLA class I molecule as described in claim 92 or 93, wherein it is an HLA-C*07:01 allele or an HLA-C*07:02 allele.

97. The HLA class I molecule as described in any one of claims 92 to 96, wherein it is a monomer.

98. The HLA class I molecule as described in any one of claims 92 to 96, wherein it is a dimer.

99. The HLA class I molecule as described in any one of claims 92 to 96, wherein it is a trimer.

100. The HLA class I molecule as described in any one of claims 92 to 96, wherein it is a tetramer.

101. The HLA class I molecule as described in any one of claims 92 to 96, wherein it is a pentamer.

102. An antigen-presenting cell (APC) comprising an HLA class I molecule as described in any one of claims 92 to 101.

103. The APC of claim 102, wherein the HLA class I molecules are expressed on the surface of the APC.

104. A method for enriching a population of target T cells obtained from a human subject, comprising contacting the T cells with an HLA class I molecule as claimed in any one of claims 92 to 101 or an APC as claimed in claim 102 or 103, wherein after the contact, the enriched population of T cells comprises a higher number of T cells capable of binding the HLA class I molecule than the number of T cells capable of binding the HLA class I molecule prior to the contact.

105. A method for enriching a population of target T cells obtained from a human subject, comprising contacting the T cells in vitro with a peptide, wherein the peptide consists of an amino acid sequence as shown in SEQ ID NO: 13, wherein after the contact, the enriched population of T cells contains a higher number of tumor-targeting T cells relative to the number of tumor-targeting T cells prior to the contact.

106. The method of claim 104 or 105, wherein the T cells obtained from the human subject are tumor-infiltrating lymphocytes (TILs).

107. A method of treating a tumor in a subject in need, comprising administering to the subject enriched T cells as described in any one of claims 104 to 106.

108. A method for enhancing cytotoxic T cell-mediated cancer cell targeting in a subject with cancer, comprising administering to the subject a peptide having an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

109. A cancer vaccine comprising a peptide having an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

110. A method for selecting T cells capable of targeting tumor cells, comprising contacting an isolated population of T cells in vitro with a peptide, wherein the peptide comprises an amino acid sequence as shown in SEQ ID NO: 97, 102, 103 or 104.

111. The method of claim 110, wherein the T cells are tumor-infiltrating lymphocytes (TILs).

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