Human T cell receptor pairs that show reactivity with the HLA-A*02:01 restricted human prostatic acid phosphatase (PAP) epitope.

Abstract

Provides a pair of human T cell receptors that exhibit reactivity with the HLA-A*02:01 restricted human prostatic acid phosphatase (PAP) epitope. [Solution] Aspects of the present disclosure relate to methods and compositions for the preparation of immune cells, including engineered T cells having a T cell receptor useful in the treatment of prostate cancer, which targets human prostatic acid phosphatase (PAP). In certain embodiments of the present invention, the T cell receptor that recognizes / binds to PAP targets at least one polypeptide epitope selected from ILLWQPIPV (SEQ ID NO: 47), TLMSAMTNL (SEQ ID NO: 48), IRSTDVDRTL (SEQ ID NO: 52), IMYSAHDTTV (SEQ ID NO: 55), KVYDPLYCESV (SEQ ID NO: 58), LLLARAASLSL (SEQ ID NO: 59), and LLFFWLDRSVLA (SEQ ID NO: 62).

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 315,825, filed on March 2, 2022, under 35 U.S.C. § 119(e), which is assigned to the assignee of the present invention and has the title "HUMAN T CELL RECEPTOR PAIRS REACTIVE WITH HLA - A*02:01 RESTRICTED HUMAN PROSTATIC ACID PHOSPHATASE (PAP) EPITOPES", and which is incorporated herein by reference in its entirety.

[0002] Statement Regarding Federal Government Funding This invention was made with government support under grant number CA009120 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Technical Field Embodiments of the present disclosure relate to at least the fields of immunology, cell biology, molecular biology, and medicine.

Background Art

[0004] Background of the Invention Prostate cancer is a disease that occurs when prostate cells mutate and begin to grow uncontrollably. Currently, prostate cancer is the second most commonly diagnosed cancer in men in developed countries worldwide and the fourth most common cause of cancer - related death.

[0005] Typical antigens that have been shown to be overexpressed by prostate cancer cells compared to their normal counterparts include proteins such as prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA). Prostate cancer cells can spread (metastasize) from the prostate to other parts of the body, particularly the bones and lymph nodes. Prostate cancer can cause pain, difficulty urinating, erectile dysfunction, and other symptoms. Typically, prostate cancer most frequently develops in men over 50 years of age, which is the most common patient population. However, in most cases, prostate cancer remains undetected, even when it is possible to determine it. The diagnosis of prostate cancer is typically made by a physical examination or a screening blood test such as a PSA (prostate-specific antigen) test. If prostate cancer is suspected, the cancer is typically confirmed by taking a prostate biopsy and examining it under a microscope. Further tests such as X-rays and bone scans may be performed to determine if the prostate cancer is spreading.

[0006] There are still unresolved challenges in treating prostate cancer. Conventional treatments such as surgery, radiation therapy, hormone therapy, chemotherapy (in some cases), proton therapy, or a combination of these can be applied to the treatment of prostate cancer. However, the man's age and underlying health status, as well as the extent of the cancer's progression, its appearance under a microscope, and the cancer's response to initial treatment are important factors in determining the disease outcome. Since prostate cancer is typically diagnosed in older men, many patients will die from other causes before the slowly progressing prostate cancer can enlarge and cause symptoms. This makes the choice of treatment difficult. The decision of whether to treat localized prostate cancer (tumor contained within the prostate) for curative purposes is a trade-off between the expected beneficial effects and adverse effects on the patient's survival and quality of life.

[0007] As is well known in this field, the immune system plays a crucial role in the treatment and prevention of many diseases. Current knowledge indicates that mammals provide various mechanisms for protecting organisms, for example, by identifying and killing tumor cells. For the purposes of this invention, these tumor cells must be detected and distinguished from the normal (healthy) cells and tissues of the organism.

[0008] The object of the present invention is to provide compositions and methods useful for the treatment of prostate cancer (PCa). [Overview of the Initiative] [Means for solving the problem]

[0009] Summary of the Invention Prostatic acid phosphatase (PAP) is a well-known prostate / prostate cancer antigen and can function as a target for cancer treatment. As described herein, HLA-A *02:01 restriction PAP epitopes were defined using multiple physical methods combined with liquid chromatography-mass spectrometry (LC-MS), including weak acid elution, immunoprecipitation, and secreted MHC IP based on the ARTEMIS platform. The recovered PAP epitopes were then used to stimulate peripheral mononuclear cells (PBMCs) from over 20 healthy donors. Reactive T cells isolated by recently developed CLInt-seq and TCR alpha / beta sequencing techniques were analyzed by 10× Genomics single-cell TCR sequencing. Polynucleotides encoding paired TCR alpha / beta chains were then introduced into normal human T cells and tested for their functionality. Using this methodology, the inventors discovered 21 TCR alpha / beta polypeptide pairs that specifically recognize and are activated by seven distinct PAP peptides. All 21 of these TCRs showed reactivity with peptide-pulsed K562-A2 cells when screened in Jurkat-NFAT-GFP cells. At least seven of these 21 TCRs can be specifically stained by their homologous tetramers. These seven candidates can successfully pair and stimulate human T cells. At least one TCR (PAP-TCR-156) exhibits significant IFNγ signaling and inhibition of target cell growth when co-cultured with cells expressing both HLA-A2 and full-length PAP. These discoveries of PAP epitopes and homologous TCR sequences are useful for novel cancer immunotherapies and vaccines.

[0010] The invention disclosed herein has several embodiments that utilize the above-mentioned findings. Embodiments of the invention include, for example, a composition of a substance comprising a polynucleotide encoding a T cell receptor (TCR) alpha chain polypeptide and / or a TCR beta chain polypeptide; where CD8 +When transduced into and expressed in T cells, alpha-chain polypeptides and / or TCR beta-chain polypeptides can form T cell receptors that recognize / bind to polypeptide epitopes on human prostatic acid phosphatase (PAP). In a typical embodiment of the present invention, polynucleotides are placed in a vector containing one or more regulatory sequences, etc., for the expression of polypeptides in cells. Embodiments of the present invention describe cells transduced with such a vector (e.g., CD8 + It also includes T cells.

[0011] In certain embodiments of the present invention, the T cell receptor that recognizes / binds to PAP targets at least one polypeptide epitope selected from ILLWQPIPV (SEQ ID NO: 47), TLMSAMTNL (SEQ ID NO: 48), IRSTDVDRTL (SEQ ID NO: 52), IMYSAHDTTV (SEQ ID NO: 55), KVYDPLYCESV (SEQ ID NO: 58), LLLARAASLSL (SEQ ID NO: 59), and LLFFWLDRSVLA (SEQ ID NO: 62). In some embodiments of the present invention, the T cell receptor that recognizes / binds to PAP targets HLA-A * 02:01 Recognizes / binds to restriction epitope. In some embodiments of the present invention, polynucleotides encoding T cell receptor (TCR) alpha chain polypeptide or TCR beta chain polypeptide (e.g., TCRs encoded by SEQ ID NOs: 1 to 42) are manipulated to introduce one or more mutations into a selected TCR polypeptide to provide the TCR with higher target killing sensitivity while typically maintaining physiological affinity (e.g., catch bond mutations). In certain embodiments of the present invention, the polynucleotide encoding T cell receptor (TCR) alpha chain polypeptide or TCR beta chain polypeptide encodes a segment of at least 5 or at least 10 amino acids of the polypeptide sequence of the alpha V region and / or the polypeptide sequence of the beta V region shown in Table A and / or Table B.

[0012] Furthermore, embodiments of the present invention include methods for inhibiting the growth of prostate cancer cells. Typically, these methods involve transducing prostate cancer cells with polynucleotides encoding T cell receptor (TCR) alpha chain polypeptide and TCR beta chain polypeptide to CD8 + This includes combining with T cells; where CD8 + When transduced into T cells and expressed, the aforementioned alpha-chain polypeptide and TCR beta-chain polypeptide can form a T cell receptor that recognizes / binds to polypeptide epitopes on human prostatic acid phosphatase (PAP). In certain embodiments of the present invention, the polynucleotide encoding the T cell receptor (TCR) alpha-chain polypeptide or TCR beta-chain polypeptide encodes the polypeptide sequence of the alpha-CDR3 region and / or the polypeptide sequence of the beta-V region shown in Table A; and / or the polynucleotide is shown in Table B. Typically, in these ways, the polynucleotide encoding the TCR alpha / beta polypeptide pair is CD8 + T cells are transduced and then combined in vivo with prostate cancer cells to treat individuals with prostate cancer.

[0013] Embodiments of the present invention further include methods for evaluating a patient's immune response to prostate cancer or prostate cancer vaccination. Typically, these methods involve observing the induction or activation of T cells obtained from a patient having prostate cancer or who has been vaccinated against prostate cancer, wherein the induction or activation of T cells is observed in response to T cell exposure to a polypeptide epitope present on human prostatic acid phosphatase (PAP); the observed induction or activation of T cells provides evidence of a patient's immune response to prostate cancer or prostate cancer vaccination. Related embodiments of the present invention also include methods for evaluating a patient's immune response to prostate cancer or prostate cancer vaccination. Typically, these methods involve observing the presence of a TCR polypeptide sequence disclosed herein or a related TCR polypeptide sequence obtained from a patient having prostate cancer or who has been vaccinated against prostate cancer, wherein the presence of a TCR polypeptide sequence disclosed herein or a related TCR polypeptide sequence provides evidence of a patient's immune response to prostate cancer or prostate cancer vaccination. A further embodiment of the present invention is a method for producing a peptide-MHC multimer composition useful for evaluating a patient's T-cell response to prostate cancer or prostate cancer vaccination, the aforementioned method comprising combining or coupling at least one polypeptide epitope present on human prostatic acid phosphatase (PAP) with an MHC multimer (e.g., an oligomeric form of an MHC molecule) so that a peptide-MHC multimer composition is produced.

[0014] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. However, while detailed descriptions and specific examples are provided for illustrative purposes and several embodiments of the present invention are shown, it should be understood that these do not limit the present invention. Many changes and modifications can be made within the scope of the present invention without departing from the intent of the present invention, and the present invention includes all such modifications. [Brief explanation of the drawing]

[0015] Brief explanation of the drawing [Figure 1] Figure 1: Flowchart and summary diagram of the entire TCR screening process project. (Left) Both monoallelic and multiallelic HLA A0201 cell lines were treated by three different physical methods for extracting peptides on MHC I. The sequences of these peptides were then identified by LC MS to obtain 27 distinct PAP epitopes. (Right) The peptides were then screened against PBMCs from more than 20 individuals, and reactive clones were identified using the CLint seq protocol (see, e.g., utility: PCT application number PCT / US20 / 49055, incorporated herein by reference).

[0016] [Figure 2] Figures 2A-2B: Schematic diagrams and data of the T2 stabilization assay designed to evaluate the stability of peptide-MHC I. Figure 2(a) Schematic diagram of the entire process of the T2 assay; Figure 2(b) Graph data showing the natural logarithmic gradient of A2 fluorescence intensity against dilution peptide concentration for various PAP peptides. Positive candidates in the T2 assay shown in Figure 2B are PAP-A2-14, PAP-A2-20, PAP-A2-21, PAP-A2-22, PAP-A2-25, and PAP-A2-27.

[0017] [Figure 3] Figure 3: Schematic diagram and data from a study using the secreted form of MHC I single-chain trimer to evaluate peptide-MHC stability. Figure 3(a) Diagram of SCT constructs; Figure 3(b) SDS-PAGE gel results of the relative yield of each PAP SCT compared to the positive control (+) WT1 peptide RMFPNAPYL.

[0018] [Figure 4]Figure 4: Schematic diagram and data from a study testing candidate TCRs in Jurkat-NFAT-GFP for rapid screening. Figure 4(a) Schematic diagram of the Jurkat-NFAT-GFP system for TCR screening. Figure 4(b) Example of FACS results for Jurkat-NFAT-GFP screening (TCR-218); top: DMSO and TCR-218 as negative control; bottom: PAP-A2-21 and TCR-218 as positive hits.

[0019] [Figure 5] Figure 5: Data from functional tests of candidate TCRs observed using various methods. Figure 5(a) IFNγ results of different TCR constructs on PBMCs using peptide dilutions; F5: positive control against MART1 peptide (EAAGIGILTV); NGFR: negative control using DMSO. Figure 5(b) IFNγ results of candidate TCR constructs on PBMCs using cell lines with and without full-length PAP; black bars: TCR-operated PBMCs and K562-A2; gray bars: TCR-operated PBMCs and K562-A2-PAP. Figure 5(c) Cytotoxicity curves of TCR-156 by incucyte using total GFP signal of target cells to quantify target cell number; blue: K562-A2 target cells and TCR-156-operated PBMCs; red: K562-A2-PAP target cells and TCR-156-operated PBMCs.

[0020] [Figure 6-1]Figures 6A-6D: Data from functional studies of TCR mutants observed using various methods. Figure 6A provides data from studies of PAP-TCR-156 mutants (see Table B), showing that the introduction of substitutional mutations can enhance the efficacy of TCRs such as PAP-TCR-156 without loss of specificity. Figure 6B provides data from studies of PAP-TCR-156 mutants that kill K62 A2 cells in the absence of PAP (left panel) and with PAP (right panel), showing that the introduction of mutations can enhance the cytotoxicity of TCRs such as PAP-TCR-156 without loss of specificity. Figure 6C provides data from studies showing PAP-TCR-156 mutant-specific cytotoxicity against prostate cancer cell lines overexpressing PAP and HLA-A2 (PC3 control cells in the left panel, PC3 cells expressing PAP in the right panel). Figure 6D provides data from studies of the PAP-TCR-156 variant, which show that these embodiments of the present invention exhibit cytotoxicity against PC3-A2-PAP with a lower E:T ratio (4:1 ratio in the left panel, 1:1 ratio in the right panel). [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0021] [Figure 7] Figure 7: Data from functional screening assays of TCR156 variants showing enhancement in PAP peptide titration assays. The left panel shows data from non-mutant TCR156(wt) and TCR156 variants 156-29, 156-30, 156-31, 156-32, 156-33, and 156-34; the right panel shows data from non-mutant TCR156(wt) and TCR156 variants 156-35, 156-36, 156-37, 156-38, and 156-39. [Modes for carrying out the invention]

[0022] Detailed description of the invention In describing the embodiments, reference can be made to the accompanying drawings, which are intended to illustrate specific embodiments that form part of the present invention and can be used to carry out the invention. It should be understood that other embodiments can be utilized and the structure can be modified without departing from the scope of the present invention. Certain aspects of the present invention disclosed below can also be found in Mao et al., Proc Natl Acad Sci USA. 2022 Aug 2;119(31) (hereinafter "Mao et al.") (the contents of which are incorporated herein by reference).

[0023] Prostatic acid phosphatase (PAP) is a well-known prostate / prostate cancer antigen and can function as a target for cancer treatment (Kantoff et al, NEJM, 2010 Jul 29;363(5):411-22). HLA-A * 02:01 restriction PAP epitopes were defined using multiple physical methods combined with liquid chromatography-mass spectrometry (LC-MS), including weak acid elution (MAE), co-immunoprecipitation (CoIP), and secreted MHC IP (sMHC-IP) based on the ARTEMIS platform (Figure 1). A total of 27 candidate PAP peptides were identified using all three methods (Table 1 in Mao et al.). The recovered PAP epitopes were then used to stimulate peripheral mononuclear cells (PBMCs) from more than 20 healthy donors. Reactive T cells isolated by recently developed CLInt-seq and TCR alpha / beta sequencing techniques were analyzed by 10× Genomics single-cell TCR sequencing (Figure 1) (Nesterenko et al, PNAS March 30, 2021). 118(13)e2100106118). Subsequently, the paired TCR alpha / beta chains were introduced into normal human T cells and tested for their functionality. Based on our previous findings (Figure 2; Tables 2,3 in Mao et al.), we discovered 21 TCR alpha / beta pairs that are specifically recognized and activated by seven distinct PAP peptides in the Jurkat-NFAT-GFP system. At least seven of these 21 TCRs can be specifically stained by their homologous tetramers. These seven candidates can be successfully paired and stimulated in human T cells. This knowledge of PAP epitopes and homologous TCR sequences may be useful in developing novel cancer immunotherapies and vaccines.

[0024] Embodiments of the present invention include compositions of substances comprising one or more vectors containing TCR polynucleotides disclosed herein. “Vector” is a composition of substances comprising isolated nucleic acids that can be used to deliver the aforementioned isolated nucleic acids into cells. Numerous vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds (e.g., polylysine compounds and liposomes) that facilitate the transfer of nucleic acids into cells. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors.

[0025] Typically, the vector is an expression vector. As used herein, the term "expression" is defined as transcription and / or translation driven by the promoter of a particular nucleotide sequence. In this context, the term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression regulatory sequence operably linked to the nucleotide sequence to be expressed. Expression vectors contain cis-acting elements sufficient for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art for incorporating recombinant polynucleotides (such as cosmids, plasmids (e.g., plasmids contained in naked form or in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus, etc.)).

[0026] Typically, the compositions of the present invention include one or more Vα / Vβ polynucleotides (e.g., polynucleotides encoding TCR Vα polypeptides combined with polynucleotides encoding TCR Vβ polypeptides such that mammalian cells (e.g., CD8 + T cells) transfected with a vector(s) can express Vα / Vβ TCRs on their surface), where the Vα / Vβ TCR recognizes a PAP peptide associated with HLA. As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0027] In another aspect, the present invention provides one or more nucleic acids encoding the TCRs disclosed herein (e.g., nucleic acids disposed within a lentiviral vector) to T cells (e.g., CD8 obtained from an individual diagnosed with cancer expressing a PAP epitope recognized by the TCR)+ The present invention includes methods for generating modified T cells, including introducing them into T cells. The present invention also includes modified T cells in which gene expression is downregulated or knocked out (for example, modified T cells having a knocked-out endogenous T cell receptor and an exogenous / transduced T cell receptor that recognizes HLA-associated PAP peptide). As used herein, the term "knockdown" refers to a reduction in the gene expression of one or more genes. As used herein, the term "knockout" refers to the removal of the gene expression of one or more genes.

[0028] The modified T cells described herein may be included in compositions for use in therapeutic regimens. The compositions may comprise a pharmaceutical composition and may further comprise a pharmaceutically acceptable carrier. A therapeutically effective dose of the pharmaceutical composition containing the modified T cells may be administered. The pharmaceutical compositions of the present invention may comprise the modified T cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or additives. Such compositions may comprise buffers (e.g., neutral buffered saline, phosphate-buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, or dextran, mannitol); proteins; polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA or glutathione); adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0029] Adoptive immunotherapy using T cells possessing antigen-specific TCRs has therapeutic potential in cancer treatment. CD8 in specific TCRs + Genetic manipulation of T cells has the advantage of redirecting T cells to selected antigens, such as PAP epitopes recognized by the TCR. In this context, in one embodiment, the present invention relates to an effective amount of modified CD8 + The method includes administering T cells to a subject to stimulate a T cell-mediated immune response against target cells or target tissue in the subject. In this embodiment, CD8 +The T cells are modified as described elsewhere in this specification. Furthermore, embodiments of the present invention involve multiple modified CD8 cells targeting multiple PAP epitopes. + This includes administering T cells. For example, embodiments of the present invention include administering at least two different modified CD8 + T cells (for example, a second CD8 that targets a PAP peptide associated with a second human leukocyte antigen) + First modified CD8 targeting the first human leukocyte antigen associated with human leukocyte antigen, combined with T cells. + This includes administering T cells.

[0030] Embodiments of the present invention are compositions of a substance comprising polynucleotides encoding T cell receptor (TCR) alpha chain polypeptide and / or TCR beta chain polypeptide, wherein the aforementioned polynucleotides are arranged in a vector and the vector is CD8 + The composition comprises a T cell receptor in which, upon transduction into a T cell, the alpha-chain polypeptide and / or TCR-beta-chain polypeptide encoded by the polynucleotide form a T cell receptor that recognizes the polypeptide epitope of human prostatic acid phosphatase (PAP). In certain embodiments of the present invention, the T cell receptor is the human leukocyte antigen HLA-A *The T cell receptor recognizes the polypeptide epitope of human prostatic acid phosphatase in combination with 02:01. In some embodiments of the present invention, the T cell receptor recognizes the polypeptide epitope of human prostatic acid phosphatase selected from ILLWQPIPV (SEQ ID NO: 47), TLMSAMTNL (SEQ ID NO: 48), IRSTDVDRTL (SEQ ID NO: 52), IMYSAHDTTV (SEQ ID NO: 55), KVYDPLYCESV (SEQ ID NO: 58), LLLARAASLSL (SEQ ID NO: 59), and LLFFWLDRSVLA (SEQ ID NO: 62). In certain embodiments of these embodiments, the polynucleotide encodes an amino acid in the TCR variable region, and the vector comprises a vector polynucleotide encoding the TCR constant region fused in-frame with the TCR variable region (see, for example, U.S. Patent Applications Publications 20220354889, 20200138865, 20210363245, and 20210155941; and Coren et al., Biotechniques. 2015 Mar 1;58(3):135-9 (which describes embodiments of the MSGV Hu acceptor vector sold by addgene®)). Typically, in these compositions, the polynucleotide is used to interact with cells (e.g., human CD8). + (T cells) are placed inside. If necessary, for example, polynucleotides are placed inside CD8 cells obtained from individuals diagnosed with cancer (e.g., prostate cancer) that express human prostatic acid phosphatase antigen. + Placed in T cells; CD8 + T cells, heterologous TCRs CD8 + Transduction is performed with a vector containing a polynucleotide encoding a TCR Vα polypeptide combined with a polynucleotide encoding a TCR Vβ polypeptide, so that the heterologous TCR is expressed on the surface of T cells, where the heterologous TCR recognizes a human prostatic acid phosphatase peptide associated with human leukocyte antigens expressed on the surface of cancer cells.

[0031] In certain compositions of the present invention, the polynucleotide encodes a segment of at least 5, 10, 25, 50, or 100 amino acids (e.g., at least 5 or 10 amino acids present in the alpha-CDR1 polypeptide sequence, alpha-CDR2 polypeptide sequence, alpha-CDR3 polypeptide sequence, beta-CDR1 polypeptide sequence, beta-CDR2 polypeptide sequence, or beta-CDR3 polypeptide sequence) of the TCR polypeptide embodiments of the present invention shown in Table A or Table B below. In certain compositions of the present invention, the polynucleotide encodes a segment of at least 100 amino acids having at least 98% sequence identity with respect to the amino acids encoded by SEQ ID NOs: 1 to 42. In some embodiments of the present invention, the T cell receptor (TCR) alpha chain polypeptide and / or TCR beta chain polypeptide encoded by the polynucleotide contains amino acid substitution mutations (e.g., SEQ ID NOs: 1 to 42) of the wild-type TCR amino acid sequence selected to optimize its interaction with its homologous ligand (e.g., Sibener et al., Cell 174, 672-687, July See 26, 2018; and Zhao et al., Science 376, 155 (2022) (the contents of which are incorporated herein by reference). In exemplary examples of such variants, the polynucleotide encodes a segment of at least 5, 10, 25, 50, or 100 amino acids, as encoded by SEQ ID NOs. 115 to 138.

[0032] Embodiments of the present invention include a method for killing cancer cells that express human prostatic acid phosphatase peptide associated with human leukocyte antigens expressed on the surface of cancer cells. For example, embodiments of the present invention include killing prostate cancer cells transduced with polynucleotides encoding T cell receptor (TCR) alpha chain polypeptide and TCR beta chain polypeptide. + This involves combining it with T cells, CD8 +The present invention includes a method for inhibiting the growth of prostate cancer cells, comprising combining alpha-chain polypeptides and TCR beta-chain polypeptides, which, when transduced and expressed in T cells, can form a T cell receptor that recognizes a polypeptide epitope on human prostatic acid phosphatase (PAP) expressed on prostate cancer cells, thereby inhibiting the growth of prostate cancer cells. In certain embodiments of these embodiments, the T cell receptor is human leukocyte antigen HLA-A * The T cell receptor recognizes the polypeptide epitope of human prostatic acid phosphatase in combination with 02:01. In some embodiments of the present invention, the T cell receptor is KELKFVTL (SEQ ID NO: 43), FQKRLHPYK (SEQ ID NO: 44), LSGLHGQDL (SEQ ID NO: 45), FQKRLHPYK (SEQ ID NO: 46), ILLWQPIPV (SEQ ID NO: 47), TLMSAMTNL (SEQ ID NO: 48), VLAKELKFV (SEQ ID NO: 49), MEQHYELGEY (SEQ ID NO: 50), GEYFVEMYYR (SEQ ID NO: 51), IRSTDVDRTL (SEQ ID NO: 52), IWSKVYDPLY (SEQ ID NO: 53), SVHNFTLPSW (SEQ ID NO: 54), IMYSAHDTTV (SEQ ID NO: 55), DFIATLGKLSG (SEQ ID NO: 56), DVYNGLLPP The methods recognize polypeptide epitopes of human prostatic acid phosphatase selected from YA (SEQ ID NO: 57), KVYDPLYCESV (SEQ ID NO: 58), LLLARAASLSL (SEQ ID NO: 59), SPIDTFPTDPIK (SEQ ID NO: 60), WQPIPVHTVPLS (SEQ ID NO: 61), LLFFWLDRSVLA (SEQ ID NO: 62), YSAHDTTVSGLQM (SEQ ID NO: 63), YSAHDTTVSGLQMA (SEQ ID NO: 64), LSELSLLSLYGIHK (SEQ ID NO: 65), IATLGKLSGLHGQD (SEQ ID NO: 66), KELKFVTLVFRHGD (SEQ ID NO: 67), and IATLGKLSGLHGQDL (SEQ ID NO: 68). In certain embodiments of these methods, CD8 +T cells are combined in vivo to treat individuals with prostate cancer. If necessary, these methods involve polynucleotides, where the polynucleotides encode a segment of at least 100 amino acids having at least 98% sequence identity with the amino acids encoded by SEQ ID NOs: 1 to 42 (as known in the art, sequence identity is expressed as a percentage of the ratio of the number of identical amino acids over the aligned length between two aligned sequences / segments).

[0033] Embodiments of the present invention include methods for evaluating a patient's immune response to prostate cancer or prostate cancer vaccination. Typically, these methods involve observing the induction or activation of T cells obtained from a patient having prostate cancer or who has received prostate cancer vaccination, where the induction or activation of T cells is observed in response to T cell exposure to a polypeptide epitope present on human prostate acid phosphatase (PAP); the observed induction or activation of T cells provides evidence of the patient's immune response to prostate cancer or prostate cancer vaccination. Optionally, in these methods, T cells express a T cell receptor that recognizes a polypeptide epitope of human prostate acid phosphatase selected from: ILLWQPIPV (SEQ ID NO: 47), TLMSAMTNL (SEQ ID NO: 48), IRSTDVDRTL (SEQ ID NO: 52), IMYSAHDTTV (SEQ ID NO: 55), KVYDPLYCESV (SEQ ID NO: 58), LLLARAASLSL (SEQ ID NO: 59), and LLFFWLDRSVLA (SEQ ID NO: 62).

[0034] Embodiments of the present invention include methods for treating a disease or condition characterized by the expression of PAP. The treatment methodology includes administering an effective amount of a pharmaceutical composition comprising the modified T cells described herein to a subject in need. The term “subject” is intended to include a living organism (e.g., a mammal) capable of eliciting an immune response. “Subject” or “patient” may be human or a non-human mammal as used herein. Non-human mammals include, for example, livestock and companion animals (mammals such as sheep, cattle, pigs, dogs, cats, and mice). Preferably, the subject is human. In a typical embodiment of the present invention, a human has cancer that expresses a PAP epitope recognized by the TCR. In some embodiments of the present invention, the cancer cells form a solid tumor. In an exemplary embodiment of the present invention, the cancer cells are prostate cancer cells.

[0035] Related embodiments of the present invention include methods for the prevention and / or treatment of individuals who have been diagnosed with cancer, are suspected of having cancer, or are at risk of developing or recurring cancer, wherein the aforementioned cancer includes cancer cells expressing a PAP epitope recognized by a TCR. The approach includes administering modified human T cells containing recombinant polynucleotides encoding a TCR to an individual, wherein the aforementioned T cells are capable of directly recognizing cancer cells expressing a PAP epitope recognized by a TCR, and the direct recognition of cancer cells includes an HLA class II restriction binding of the TCR to the PAP epitope recognized by the TCR expressed by the cancer cell.

[0036] The operated CD8 of the present invention + Regarding the use of T cells, the method generally involves administering an effective amount (e.g., by intravenous or intraperitoneal injection) of CD8 +This includes administering a composition containing T cells to an individual in need. A suitable pharmaceutical composition can be adapted for administration by any suitable route (such as parenteral (including subcutaneous, intramuscular, or intravenous), intestinal (including oral or rectal), inhalation, or intranasal route). Such compositions can be prepared by any method known in the pharmaceutical field (e.g., mixing of the active ingredient with carriers or additives under sterile conditions).

[0037] In another aspect, the present invention relates to the manufacture of a pharmaceutical for the aforementioned treatment in subjects requiring treatment of a disease or condition characterized by PAP expression, using the polynucleotides or modified CD8 described herein. + This includes the use of T cells. In exemplary embodiments of the present invention, the disease is a cancer (e.g., prostate cancer) that expresses the PAP epitope disclosed herein.

[0038] The technologies in this domain have been properly developed, and several methods and materials known in the art can be adapted for use with the inventions disclosed herein. Such methods and materials are disclosed, for example, in U.S. Patent Applications Publications 20190247432, 20190119350, 20190002523, 20190002522, 20180371050, 20180057560, 20170029483, 20160024174, and 20150141347 (the contents of which are incorporated herein by reference).

[0039] Certain aspects of the present invention are disclosed in Mao et al. Proc Natl Acad Sci USA.2022 Aug 2;119(31):e2203410119.doi:10.1073 / pnas.220341011 (both of which are incorporated herein by reference). All publications mentioned herein (e.g., Zah et al., Nature Communications volume 11, Article number: 2283 (2020) and international patent applications PCT / US19 / 49484, WO2021 / 046121, and PCT / US2020 / 037486, as well as Kantoff, Phillips, et al. "Sipuleucel-T Immunotherapy for Castration-Resistant Prostate Cancer." N Engl J Med 2010;363:411-422; Fong, Lawrence, et al. "Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy." J Immunol 2001;167(12)7150-7156; and Nesterenko, Pavlo, et al. "Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific Publications disclosed herein, such as "TCR cloning" (PNAS 2021;118(3)), are incorporated herein by reference to disclose and describe aspects, methods, and / or materials relating to the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art. Unless otherwise defined, all technical terms, notations, and other scientific terms or glossaries used herein are intended to have meanings commonly understood by those skilled in the art to which the invention pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and such definitions included herein should not necessarily be construed as representing a substantial difference beyond the common understanding in the art. [Examples]

[0040] Example 1: Physical and in silico immunopeptide profiling of the cancer antigen prostatic acid phosphatase reveals targets from which TCRs can be isolated. Certain aspects of the present invention disclosed below are based on Mao et al., Proc Natl. This can be found in Acad Sci USA. 2022 Aug 2;119(31) (hereafter referred to as "Mao et al.") (its contents are cited as reference).

[0041] Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Tumor recognition by T cells is mediated by interactions between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Elucidating the identity of MHC-bound peptides is crucial for discovering homologous TCRs and predicting potential toxicity. We performed a multi-form immunopeptide-mediated analysis against prostatic acid phosphatase (PAP) (a well-recognized tissue antigen). Using three physical methods, including weak acid elution, immunocoprecipitation, and secretory MHC precipitation, we analyzed HLA-A *At 02:01, a thorough signature of PAP was captured. A total of 27 PAP peptides were identified, but the commonly used algorithm NetMHCpan4.0 predicted only 5 of these peptides. Peripheral blood mononuclear cells (PBMCs) from over 20 healthy donors were screened using PAP peptides. Twenty-one congeneral TCRs for seven distinct epitopes were identified using single-cell isolation techniques that detect intracellular IFNγ and TNFα. One TCR contained full-length PAP and HLA-A * The cell lines expressing both 02:01 show reactivity. Our results demonstrate that a combination of diverse immunopeptidomic approaches is productive for elucidating target peptides and defining the first cloned TCR sequence for prostatic acid phosphatase.

[0042] Prostatic acid phosphatase (PAP) is a target for prostate cancer immunotherapy. One way to prevent "on-target off-tumor" toxicity is to select tissue antigens expressed on non-essential organs. Patients with advanced prostate cancer often undergo radical prostatectomy to remove the prostate (11). The inventors selected prostatic acid phosphatase (PAP) from among a number of previously defined prostate tissue antigens because: 1. PAP expression is restricted to the prostate and prostate cancer (12); 2. PAP expression can be found in more than 95% of prostate cancers (13); 3. Elevated serum PAP has been found in more than 60% of patients with recurrent prostate cancer (14); and 4. The secretory form of PAP does not compete with TCR-PAP recognition because its interaction is restricted to MHC I-bound peptides.

[0043] Prior attempts to target PAP led to cyproisel-T (Provenge), the first cancer vaccine to receive FDA approval (15). Clinical trials showed a median improvement of 4.1 months in overall survival in men with metastatic castration-resistant prostate cancer (15). The T cell proliferation response in vaccinated individuals was measured by the stimulation index (SI) (15). SI is the ratio of T cells cultured with an antigen. 3 It is defined as the ratio of H-thymidine inclusion to the control group (15). A positive T cell proliferation response was defined as an SI greater than 5 measured at 6 weeks post-immunization (15). Among patients treated with Provenge, 27.2% showed a response to PAP (compared to 8.0% in placebo) (15). In addition, recent studies have shown that T cells from patients treated with cyproisel-T cells respond to PAP. + Video evidence was provided showing that target cells can be lysed (16). Neither the presented PAP epitope nor the homologous TCR sequence is defined at the molecular level. By recovering TCRs that specifically recognize the PAP epitope, products for potential therapeutic treatments can be obtained.

[0044] In silico prediction of epitopes on MHC I is an important strategy, but not sufficient. Multiple computational methods have been developed to predict peptide-MHC binding affinity using knowledge based on experimentally defined epitopes (17-19). In silico prediction is widely used because it can provide results quickly (18). Previous attempts to identify PAP epitopes also relied primarily on motif-based predictions (20, 21). In a recent study, Wells et al. assembled a consortium (TESLA) containing 25 different comparison platforms (22). pMHC multimer staining found that only 6% of the predicted peptides were immunogenic (22). Finding consensus among in silico pipelines is also difficult. Overlap between different algorithms in TESLA is limited: between any two randomly selected methods, only 13% was found in the median and 62% in maximum (22). Defining MHC I-bound peptides solely by prediction is difficult for two reasons: 1. the selection of a single and best tool to use, and 2. the evaluation of false positives and false negatives of prediction results.

[0045] Use of physical assays to define the immunopeptideome An alternative method for defining the immunopeptideme is to directly isolate MHC I-bound peptides and identify them by liquid chromatography and mass spectrometry (LC-MS). Several physical methods using mass spectrometry to define the immunopeptideme have been previously developed (including weak acid elution (MAE), MHC co-precipitation (CoIP), and secreted MHC immunoprecipitation (sMHC-IP)). MAE was one of the earliest approaches to isolating peptides from MHC I by using isotonic buffer to destabilize the peptide-MHC complex (23). While rapid and convenient, this method can result in non-MHC-bound peptides from other extracellular proteins. CoIP purifies the peptide-MHC I complex using monoclonal antibodies to obtain results with less non-MHC peptide contamination (24, 25). This requires a large amount of antibody as well as the expression of both the target antigen and the desired HLA type on target cells. The sMHC-IP technique requires the manipulation and expression of soluble single-chain MHC in cell lines for affinity capture (26, 27). This protocol requires cell line manipulation and may generate peptides that can only be presented on artificial constructs. There is no consensus on a single best approach. To capture a more comprehensive immunopeptide signature of PAP, we have developed an HLA-A protocol. * A combination of all three approaches above for 02:01 (one of the most common subtypes) (28).

[0046] Identification of TCRs derived from antigen-reactive single T cells To date, no single-cell MHC I restriction TCR sequences for the PAP epitope have been identified or disclosed in any publicly available immunoepitope database (IEDB) (29). One of the main challenges has been enriching and identifying congeneral T cells for single-cell sequencing. We have recently developed a technique called CLint-seq that can isolate single activated T cells. Cells were fixed with a disulfide bond-based reversible crosslinking agent (DSP) and sorted based on intracellular activation markers by fluorescent cell sorting (FACS) (30). The use of a reversible crosslinking agent makes it possible to release cellular mRNA from the mRNA-protein crosslinking complex. These mRNAs can then be efficiently reverse transcribed and meet the quality requirements for the 10× Genomics single-cell TCR sequencing platform (30, 31). T cells stimulated by congeneral peptides can produce cytokines (such as IFNγ and IFNα) that can be captured and intracellularly stained. Using physically determined PAP epitopes, 21 peptide-reactive TCRs were successfully isolated from healthy donor PBMCs using CLint-seq.

[0047] result: HLA-A * 02:01 Immunopeptide profiling of diverse PAP formulas shown above Using both physical and in silico approaches, we thoroughly investigate HLA-A in PAP. * 02:01 The immunopeptide-dominant signature was defined. The commonly used algorithm NetMHCpan4.0 was applied to HLA-A * PAP epitopes above 02:01 were profiled (18). Forty PAP peptides were profiled using the top two percentiles as cutoffs for HLA-A * It was selected as a potentially good binder at 02:01 (Supplementary Table 1 in Mao et al.).

[0048] To determine the presence of these predicted peptides and others, three previously published physical methods (including weak acid elution (MAE), co-immunoprecipitation (CoIP), and secretory MHC immunoprecipitation (sMHC-IP)) were performed (Figure 1). The MAE protocol uses an acidic buffer (pH 3.3) to dissociate the peptide-MHC I complex. This protocol was used for HLA-A of single allele (K562-A2-PAP) and multi-allele (M202-PAP). * 02:01 + The strategy was applied to both cell lines. Since wild-type K562 cells lack surface MHC I, K562-A2-PAP is considered a single HLA allele cell line (32). This strategy identified a total of 11 PAP peptides (Supplementary Figure 1a in Mao et al.; Table 1).

[0049] Since treatment with MAE can induce the release of non-MHC peptides, an alternative approach, CoIP, was performed on the same two cell lines. This approach uses a monoclonal antibody (clone W6 / 32) to enrich MHC I released from the cell surface after lysis (24, 25, 33). The MHC I-bound peptides are then dissociated from the purified product and analyzed by LC-MS / MS. Twelve PAP peptides were recovered by CoIP (Supplementary Figure 1a in Mao et al.; Table 1). Two of the peptides overlapped with those found by MAE (Supplementary Figure 1a in Mao et al.; Table 1).

[0050] Secreted MHC-IPs (sMHC-IPs) were previously developed to enhance the higher expression of MHC I in its engineered soluble form as single-chain dimers (SCDs) (26, 27). The recently released sMHC-IP platform ARTEMIS enhances the expression of soluble HLA-A *Robust expression and secretion of the 02:01 molecule are achieved (27). This manipulated form includes a hexahistidine tag (6×His tag) to enhance enrichment efficacy with Ni-NTA agarose. Eight peptides, including six not found by the other two physical methods, were recovered by sMHC-IP (Supplementary Figure 1a in Mao et al., Table 1). No single peptides were found by any of the three methods (Supplementary Figure 1a in Mao et al.; Table 1). A comparison between the physical and in silico approaches showed only five peptide overlaps, which accounted for 12.5% ​​of the total peptides predicted by NetMHCpan4.0 (Supplementary Figure 1b in Mao et al.; Table 1).

[0051] Next, BLAST analysis was performed on all physically recovered PAP epitopes in the human protein library to test their specificity to PAP (34). All 27 PAP peptides were specific to the PAP sequence. Peptides with similar sequences were mostly derived from other members of the acid phosphatase family, such as lysosomal acid phosphatases and testicular acid phosphatases (Mao et al.). (Supplemental table 2 in al.)

[0052] HLA-A of recovered PAP peptides * 02:01 Evaluation of specificity Some of the peptides recovered by physical methods were non-HLA-A * 02:01 subtype or peptide fragments not located on MHC I may be derived from the recovered PAP epitope. * To evaluate 02:01 specificity, a T2 cell binding assay was performed. T2 cell lines lack antigen processing-associated transporter (TAP) proteins responsible for peptide loading onto MHC I. As a result, a limited amount of unstable MHC I (HLA-A) was obtained. *Only molecules (including 02:01) are spontaneously presented on T2 cells (35). In the T2 binding assay, chemically synthesized candidate peptides (purity over 80%) are exogenously added to the growth medium. * Epitopes with 02:01 specificity can form stable peptide-MHC complexes, inducing the accumulation of these molecules. Subsequently, HLA-A * The 02:01 quantity can be quantified using a flow cytometer with an anti-A2 antibody (clone BB7.2) conjugated to FITC (Figure 2a).

[0053] Twenty-seven PAP peptides, defined by physical methods, were tested in a T2 binding assay. Six of the 27 PAP peptides showed high HLA-A2 signaling when exogenously pulsed in T2 cells (Table 1, Figure 2b in Mao et al.). All six peptides could be detected by sMHC-IP (including one epitope found by both sMHC-IP and CoIP). Five of these six peptides showed potent HLA-A2 signaling. * The 02:01 binder passed the 2% selective cutoff of NetMHCpan4.0 (Table 1 in Mao et al.).

[0054] Peptide-MHC I complexes processed by endogenous mechanisms may exhibit different stability compared to exogenous peptide pulsing (such as in T2 binding assays). This may be a result of post-translational modification (PTM). The relative stability of target pMHCs was evaluated using secreted monochain trimers (SCTs), a recently developed technique (36). In secreted SCT constructs, the MHC I heavy chain (HLA-A with H74L and Y84C mutations) * 02:01 The alpha chain, light chain (beta-microglobulin), and corresponding peptide were linked together as a single-chain molecule by a linker (Figure 3a). The construct was expressed in cells and released into the culture supernatant. HLA-A *Only peptides favored by 02:01 are expected to produce stable SCTs and have a higher yield. The amount of SCTs will be measured by band intensity in SDS-PAGE, and HLA-A * The data was normalized to the well-known WT1 cancer epitope (RMFPNAPYL) restricted to above 02:01 (Figure 3b) (37, 38).

[0055] The nine peptides were HLA-A when using a normalized cutoff of 0.2 against the control WT1 epitope. * Relatively high stability was found on 02:01 (Table 1 in Mao et al.; Figure 3b). Five of these nine PAP peptides were not among the candidates selected by NetMHCpan4.0 (top 2%) (Table 1 in Mao et al.). Only four of these nine peptides scored positively in the T2 binding assay, suggesting that each evaluation technique can address different aspects of peptide-MHC stability (Table 1 in Mao et al.). In particular, PAP_A2_24 showed a higher yield (1.40) than the positive control, but its predictive score by NetMHCpan4.0 was poor (24.22%) (Table 1 in Mao et al.; Figure 3b).

[0056] Post-translational modified PAP peptides are HLA-A * The binding affinity to 02:01 increases. PAP-A2-24 is HLA-A in different stability assays. *The 02:01 binding yields contradictory results. One possible explanation is that PAP-A2-24 is post-translationally modified. Previous literature has reported N-glycosylation of PAP-A2-24 to asparagine (N220 of PAP) (39). To investigate whether an N-glycosylated form of PAP-A2-24 is presented, both SCT products of PAP-A2-24 (SVHNFTLPSW (SEQ ID NO: 54)) and PAP-A2-25 (IMYSAHDTTV (SEQ ID NO: 55)) were treated with PNGase F, which can specifically remove N-glycans (40). SDS-PAGE results show that the PAP-A2-24 SCT showed a band with an apparently higher molecular weight than PAP-A2-25 before PNGase F treatment. Both SCTs migrate similar distances in the gel after deglycosylation (Supplementary Figure 2a in Mao et al.). These results indicate the presence of additional N-glycans on PAP-A2-24, in addition to the glycosylation sites on the heavy and light chains of MHC I. The only sequence difference between PAP-A2-24 SCT and PAP-A2-25 SCT is present in these peptide fragments. It is highly probable that the additional N-glycans were present within the peptide (SVHNFTLPSW (SEQ ID NO: 54)).

[0057] Furthermore, the spectrum was re-analyzed to confirm the presence of the deglycosylated form, PAP-A2-24. Previous reports suggest that N-glycosylated asparagine (N) can be enzymatically deamidated to aspartic acid (D) (41). Both of the following forms were detected by LC-MS in the CoIP results: SVHNFTLPSW (SEQ ID NO: 54) and SVHDFTLPSW (SEQ ID NO: 69) (Supplementary Figure 2b in Mao et al.).

[0058] Isolation of PAP peptide-specific TCRs from PBMCs of healthy individuals PBMC cells recovered from multiple commercially available normal donors (n>20) were screened to identify TCRs responsive to PAP peptides. T27 chemically synthesized peptides were added to total PBMCs containing a mixture of antigen-presenting cells (e.g., monocytes and B cells) capable of priming T cells. T cells were then cultured and grown for 10 days. The CLint-seq protocol was then applied to stimulated cells to isolate candidate reactive T cells (30). As discussed above, TNFα + / IFNγ + Fixed CD8 The T cell population was enriched with reactive cells by FACS selection. TCR pairs that appeared more than once (frequency greater than 1) as a result of 10× Genomics sequencing were selected as potential PAP-reactive clones. 124 candidate α / β pairs were collected from 8 healthy individuals (including 3 females, 4 males, and 1 of unknown gender) (Supplementary Table 3 in Mao et al.).

[0059] To determine whether these TCRs were responsive to PAP, the alpha and beta TCR variable regions from all selected candidates were then synthesized into DNA fragments for cloning. To reduce mispairing with endogenous human TCRs, the constant regions (TRAC and TRBC) of both the alpha and beta chains were replaced with mouse constant regions. To ensure equal expression, the alpha and beta chains of the paired TCRs were linked to a mutated autocleaved 2A peptide linker (F2Aopt) (42).

[0060] Next, the manipulated TCR sequence was cloned into a pMAX-cloning vector for rapid functional screening using electroporation. The pMAX construct containing the target TCR was electroporated into the Jurkat-CD8-NFAT-GFP cell line, which was used as a reporter system. In Jurkat-CD8-NFAT-GFP cells, GFP expression is induced by binding and activation of the NFAT promoter repeat after TCR activation (Figure 4a). GFP expression can then be quantified by flow cytometry to determine whether the TCR recognized the homologous peptide-MHC I. The mouse TCR beta chain was measured by FACS to estimate transfection efficiency. K562 cells were transfected with lentivirus to HLA-A * Transduction was performed with 02:01-IRES-GFP (K562-A2), and used as the target cell during the study (Methods). Individually chemosynthesized PAP peptides were added to K562-A2 cells and presented by these cells. Effector cells (Jurkat) and target cells (K562) were mixed in a 2:1 ratio. From 124 candidate clones, 21 TCRs were found to recognize seven distinct PAP peptides previously defined by LC-MS (Table 1 in Mao et al.; Supplementary Table 4). These 21 TCRs were derived from three individuals, including two males and one female (Supplementary Tables 3,4 in Mao et al.).

[0061] Verification of candidate TCR function in human PBMCs Next, 21 candidate TCRs that showed reactivity in the Jurkat-CD8-NFAT-GFP system were tested in human PBMC cells. Selected TCR constructs with a mouse constant region were tracked using a truncated low-affinity neuron growth factor receptor (delta-LNGFR) as a transduction marker. Candidate TCRs were transduced into human PBMCs using the pMSGV retrovirus system (9) (Methods). Transduction efficiency was estimated by measuring surface dLNGFR levels by FACS. Mouse TCR beta chains were also quantified by FACS to evaluate whether the TCRs were transported to the cell surface. Tetramers containing the individual PAP peptides of interest were produced and used against the engineered PBMCs to ensure specific recognition (Supplementary Figure 3 in Mao et al.).

[0062] Stimulated T cells that recognize congener peptides bound to MHC I can release cytokines such as IFNγ. IFNγ was quantified by performing ELISA using recombinant IFNγ as a standard (Methods). Individual PAP peptides were exogenously added to K562-A2 cells. Manipulated PBMCs and target K562-A2 cells were mixed in a 2:1 (effector:target) ratio. The supernatant from the co-culture experiment was then collected after 48 hours. Seven TCRs showed significant IFNγ signaling for three distinct PAP peptides when expressed in human PBMCs (Figure 5a in Mao et al.; Table 1). Notably, five of these seven TCRs were for a peptide (PAP-21) that did not pass the predicted cutoff (<2%) using the NetMHCpan4.0 algorithm (Table 1 in Mao et al.).

[0063] TCRs showing high IFNγ signaling in PBMCs were tested using serial dilutions of congeneral peptides, and their relative potency was compared to the clinically tested TCR, F5. This TCR against the MART1 epitope (EAAGIGILTV) was previously isolated from melanoma patients (6). The F5 TCR can induce tumor regression in patients without affinity maturation to increase its potency and served as a control in our experiments (6). Chemically synthesized peptides were tested against K562-A2 at various concentrations. PBMCs expressing candidate PAP TCRs were mixed in a 2:1 (effector:target) ratio. IFNγ ELISA was performed on the supernatant collected after 48 hours. In particular, one PAP TCR (PAP-TCR-204) showed similar activation levels with peptide dilutions compared to F5, while the remaining six TCRs showed weaker results (Figure 5a).

[0064] Next, PBMCs expressing these seven TCRs were co-cultured with target cells expressing full-length PAP to test their ability to recognize processed PAP epitopes. Full-length PAP isoform 2 (TM-PAP) was transduced into K562-A2 cell lines using lentivirus. The transduced population was single-cell sorted and expanded to create clonal cell lines strongly expressing PAP. TCR-modified PBMCs were mixed with target K562-A2-PAP cells in a 16:1 (effector:target) ratio. PBMCs transduced with F5 TCR and dLNGFR only (no TCR) empty vectors were used as negative controls. ELISA was performed on the co-culture supernatant after 48 hours. One TCR (PAP-TCR-156) showed specific recognition of full-length PAP and produced 20,000 pg / ml of IFNγ (Figure 5b). Four TCRs (128, 215-1, 218, and 219) produced low IFNγ signals (approximately 3,000 pg / ml) (Figure 5b).

[0065] Cytotoxicity of candidate PAP TCRs was evaluated by recording total target viable cells using the IncuCyte platform. Target K562-A2-PAP cells co-expressed GFP, and GFP was identified by real-time imaging and analysis. - It can be distinguished from PBMC cells. Live cells were imaged every 2 hours, and the number of target cells was recorded over a 120-hour period. The GFP signal was then processed using the IncuCyte analysis tool to estimate the target cell region. One of the candidate TCRs, PAP-TCR-156, can inhibit the growth of cells expressing full-length PAP (Figure 5c). The total GFP region of K562-A2-PAP was maintained at similar levels during 150 hours of co-culture with PBMCs expressing PAP-TCR-156 (Figure 5c). The total GFP region for K562-A2 cells showed a 3-fold increase as a negative control (Figure 5c).

[0066] Materials and methods Weak acid elution: A weak acid elution protocol (53) for eluting MHC I-associated peptides, mainly based on a previously published protocol with some modifications. 1-2 × 10 8A total of 100 cells were used. M202-PAP cells were dissociated using 1×PBS + 1 mM EDTA, while K562-A2-PAP cells were collected by spinning down at 1500 RPM for 5 minutes. The target cells were then washed three times with 1×HBSS buffer (Thermo Fisher). 25 ml of weak acid elution buffer (0.131 M citrate, 0.066 M Na2HPO4, 150 mM NaCl, 0.3 μM aprotinin, 5 mM iodoacetamide, pH 3.3) was applied to the target cells and gently shaken at room temperature for 2 minutes. The samples were then spun at 4000×g for 5 minutes at 4°C, and the supernatant was collected. Formic acid was added to the samples until a final concentration of 0.1% (v / v) was reached. A 3 ml C18 solid-phase extraction cartridge (3M) was pre-rinsed three times with 99.9% acetonitrile (ACN) + 0.1% formic acid. The MAE sample was then added to the C18 column and washed three times with 0.1% formic acid in water. The C18 column was then eluted three times with 200 μl of 40% ACN + 5% formic acid + 55% H2O. The sample was then passed through a 3 kD centrifuge filter (Millipore) at 4°C at 4000 × g for 90 minutes. The flow-through was then dried by vacuum centrifugation and stored at -20°C until MS analysis.

[0067] MHC I CoIP: The CoIP protocol was modified based on previously published procedures (54, 55). 1-2 × 10 8 Individual M202-PAP cells or K562-A2 PAP cells were harvested by either a non-enzymatic dissociation reagent (1×PBS + 1 mM EDTA) or by spin-down at 1500 rpm for 5 minutes. The cells were first washed three times with 1×PBS. Then, CoIP lysis buffer (20 mM Tris (pH 8.0), 1 mM EDTA, 100 mM NaCl, 1% Triton® X-100, 60 mM n-octyl glucoside, 1 mM PMSF (Sigma-Aldrich), protease inhibitor (Roche Life Science), and 1 mg / ml DNase I (Roche Life Science) was added. 7Cells were lysed using 1 ml of lysis buffer per cell. The sample was then shaken at 4°C for 1 hour. Next, the lysate was centrifuged at 10000 × g for 20 minutes to pelletize the debris. The supernatant was then 10 8 GammaBind Plus Sepharose beads (GE Lifesciences) conjugated with W6 / 32 antibody (BioXCell) were combined at a ratio of 1 ml of beads per cell. The bead-lysate mixture was shaken at 4°C for 180 minutes. The mixture was then loaded onto a Poly-Prep chromatography column (Bio-Rad). The column was then washed four times with 10 ml of Wash Buffer I (CoIP Wash Buffer I: 20 mM Tris (pH 8.0), 1 mM EDTA, 100 mM NaCl, 60 mM n-octyl glucoside, and 1 mg / ml DNase I), four times with 10 ml of Wash Buffer II (CoIP Wash Buffer II: 10 mM Tris (pH 8.0)), and once with 10 ml of ultrapure water (Thermo Fisher). The peptide was released from the beads for 2 minutes by adding 10% acetic acid (Sigma), and then cleaned up by spinning at 3000 × g for 30 seconds using a 0.45 μm Costar Spin-X centrifuge tube filter (Corning). The sample was then flash-frozen and stored at -70°C until further processing.

[0068] Secreted MHC-IP using the ARTEMIS protocol: The ARTEMIS protocol was based on a previously published protocol (27). Secreted expression of both HLA-A2 and PAP was achieved using a lentiviral transduction system in free-style 293-F cells (Thermo Fisher). 400 ml of supernatant containing secreted MHC I was purified with Ni-NTA agarose (1 μL slurry per 1 ml supernatant). The slurry was loaded onto a Poly-Prep chromatography column and washed. The denatured samples were stored at -70°C until further processing.

[0069] LC-MS analysis: The eluted sample was loaded onto a HyperSep C18 column (Thermo Scientific 60108-390), washed three times with 0.1% formic acid, and then eluted with elution buffer (40% acetonitrile, 0.1% formic acid). The desalted sample was freeze-dried under rapid reduced pressure and then reconstituted with water. Next, the sample was treated with a surfactant removal kit to remove any remaining surfactant from the lysis buffer. Finally, the sample was acidified to contain 5% formic acid before loading into LC-MS. The sample was delivered to an Orbitrap Fusion Lumos hybrid mass spectrometer using a 140-minute gradient (0-5 min, 1-5.5% B; 5-128 min, 5.5-27.5% B; 128-135 min, 27.5-35% B; 135-136 min, 35-80% B; 136-138 min, 80% B; 138-138.5 min, 80-1% B; 138.5-140 min, 1% B; B: 80% CAN + 0.1% formic acid). Acquisition was performed under the following data-dependent acquisition (DDA) mode: a full MS scan was acquired at a resolution of 120K using an Orbitrap mass spectrometer, and monovalent and polyvalent ions above 800 m / z were selected to be fragmented using high-energy collision dissociation (HCD) at 32% collision energy. Subsequently, an MS / MS scan was performed at a resolution of 15K using the Orbitrap. Dynamic exclusion prevented repeated selection of ions with the same m / z within a 60-second period. Data retrieval was performed using the Crux pipeline (v3.2) on the non-specifically digested EMBL human reference proteome (UP000005640human_9606), and PSM and peptide FDR were set to a 1% threshold.

[0070] T2 peptide binding assay: T2 cells (ATCC) were cultured in IMDM (Thermo Fisher) containing 20% ​​FBS (Omega Scientific). Before peptide loading, 2 × 10⁶ cells were cultured. 5Cells were resuspended in 100 µl of serum-free RPMI (Thermo Fisher) and added to each well of a 96 µl bottom tissue culture plate (Corning). Chemosynthesized peptides were diluted to multiple concentrations in serum-free RPMI and added to designated wells containing T2 cells. Cells containing the peptides were co-cultured overnight in an incubator at 37°C. The cells were then washed twice with 1 × PBS and stained with 2 µl of anti-HLA-A2 FITC antibody (clone BB7.2, Biolegend) per well. The amount of HLA-A2 molecules was quantified by FACS.

[0071] SCT quantitative assay: SCT constructs containing individual PAP peptides (mutant H74L / Y84C) were synthesized according to previously published protocols (36).

[0072] Cell culture: K562 (ATCC), M202 (provided by A. Ribas of UCLA), and Jurkat-NFAT-ZsGreen (provided by D. Baltimore of Caltech) were cultured in RPMI1640 (Thermo Fisher) containing 10% FBS (Omega Scientific) and glutamine (Fisher Scientific). 293T (ATCC) was cultured in DMEM (Thermo Fisher) containing 10% FBS and glutamine. Naive peripheral blood mononuclear cells (PBMCs) for stimulation were cultured in TCRPMI containing 50 U / ml IL-2 (Peprotech) and the target PAP peptide (purity >80%, Elim Biopharm) chemically synthesized as previously described (P Nesterenko, Cell Reports, 2021). TCRPMI medium contains: RPMI1640 (Thermo Fisher), 10% FBS (Omega Scientific), Glutamax (Thermo Fisher), 10 mM HEPES (Thermo Fisher), non-essential amino acids (Thermo Fisher), sodium pyruvate (Thermo Fisher), and 50 μM β-mercaptoethanol (Sigma). PBMCs for retroviral transduction were first activated with CD3 / CD28 dynabeads (Thermo Fisher) and cultured in the following T cell medium (TCM): AIM V medium (Thermo Fisher), 5% human AB serum (Omega Scientific), 50 U / ml IL-2 (Peprotech), 0.5 ng / ml IL-15 (Peprotech), Glutamax (Thermo Fisher), and 50 μM β-mercaptoethanol (Sigma).

[0073] CLInt-seq: Reactive T cells were isolated from stimulated PBMCs using CLInt-seq according to a previously published protocol (30). After co-culturing with a PAP peptide pool for 7-10 days, the PBMCs were transferred to 96-well U plates and left to stand overnight. The cells were then cultured for 1 hour with a 10 ug / ml peptide pool and 1 ug / ml CD28 / 49d antibody (BD Biosciences), followed by the addition of brefelzin A (Biolegend). After incubation at 37°C for approximately 8 hours, the cells were treated as previously described and analyzed by FACS for CD3 + / CD4 - / CD8 + / TNFα + / IFNγ + The group was stained (56).

[0074] Single-cell TCR sequencing: CD8+ T cells producing both TNFα and IFNγ were sorted into approximately 30 µl of 0.04% BSA solution. When fewer than 1000 cells were isolated, 5000–10000 K562 cells would be sorted into the same tube as a carrier population. A 10× Genomics single-cell TCR V(D)J library was then constructed using the UCLA Technology Center for Genomics & Bioinformatics. TCR pairs were then sequenced using MiSeq (Illumina).

[0075] Jurkat-NFAT-GFP assay: As previously described, candidate TCRs were rapidly screened in Jurkat-NFAT-GFP cells (56).

[0076] Transduction of TCRs in PBMCs: Candidate TCRs in PBMCs were manipulated according to previous publications (56).

[0077] Preparation of MHC tetramers: MHC tetramers used to stain candidate PAP TCRs were synthesized and prepared according to previously published protocols (57).

[0078] T cell activation analysis: For peptide pulsing co-culture experiments, target cells were mixed with TCR-modified PBMCs in a 1:2 (T:E) ratio in medium supplemented with 1 μg / ml anti-CD28 / CD49d antibody (BD Biosciences), which is preferable for target cells. For cell lines expressing full-length PAP, target cells were initially treated with 2 ng / ml IFNγ and 3 ng / ml TNFα for 8–10 hours. Subsequently, target cells and PBMCs were mixed in a 1:16 (T:E) ratio for co-culture analysis. The supernatant was collected after 48 hours and analyzed by ELISA (BD Biosciences) to estimate the IFNγ concentration.

[0079] Cytotoxicity analysis using IncuCyte: Target cells were plated onto 96-well tissue culture plates coated with 0.001% poly-L-lysine (Sigma) and held at 37°C for approximately 2 hours. Then, TCR-modified PBMCs were added to the desired wells in an effector:target ratio of 2:1 (peptide-pulsed target cells) or 16:1 (full-length PAP target cells). Plates containing the cell mixture were analyzed using the IncuCyte system for 120 hours using the GFP surface region to estimate T cell death.

[0080] Consideration Our research using multiple immunopeptide approaches identified 27 potential HLA-A * 02:01 Identifying restriction PAP peptides. The inventors were able to recover 21 candidate PAP TCRs for 7 of the defined epitopes. The 7 TCRs reacted to 3 distinct epitopes on target cells pulsed with the peptide when manipulated in human PBMC cells. Of these, one TCR (PAP-TCR-156) was found to be full-length PAP and HLA-A * 02:01 It can recognize peptides presented on cell lines expressing alleles.

[0081] All three physical assays (MAE, CoIP, and sMHC-IP) were able to generate immunogenic peptides. For TCRs that act efficiently in PBMCs, all three homologous peptides can be detected by sMHC-IP. Despite the relatively small size of our samples, sMHC-IP appears to be the most efficient method for recovering immunogenic epitopes.

[0082] Reagents for PAP-specific T cells can be developed using epitopes defined by physical methods. The peptide of interest can be refolded into MHC-based multimers for use as detection and isolation reagents. A common form in which four peptide-MHC molecules are attached to a streptavidin molecule is called a "tetramer." More complex versions of the multimer have also been available by adding further fluorophores and increasing the number of MHC monomers (e.g., pentamers or dextramers) (43). The production of MHC multimers largely depends on knowing the identity of the peptide. Seven of our PAP TCRs recovered against three distinct PAP peptides can be specifically stained by their homologous tetramers. Other candidates in our list may also be used in the production of multimers. These reagents may be useful in pre-screening for PAP-responsive T cells from patients or healthy donors being treated in Provenge.

[0083] One of the PAP epitopes (PAP-A2-24) is a glycosylated HLA-A * It showed altered affinity for 02:01. Both the undenatured and deglycosylated (N to D) forms were detected by LC-MS. Since cancer can produce abnormal carbohydrate modifications on proteins, post-translational modifications such as glycosylation may generate a larger pool of epitopes for immunotherapy (44, 45).

[0084] One of our candidate TCRs, PAP-TCR-156, shows potential to recognize cell lines expressing full-length PAP. The qualities of this TCR indicate a weak T-cell response as measured by IFNγ and cytotoxicity assays. Increased potency of these candidate TCRs is necessary for future applications and trials.

[0085] One way to enhance the sensitivity and potency of T cells is to increase their affinity for the TCR (a process called "TCR affinity maturation") (46). Previous results have shown that higher affinity can lead to a more rapid and potent response (47). Common methods for TCR affinity maturation include: 1. untargeted mutagenesis, 2. site-directed mutagenesis, and 3. single-amino acid (AA) screening of the TCR complementarity-determining region (CDR) (7, 48, 49).

[0086] Furthermore, more potent TCRs can be obtained by using alternative sources of T cells. We used PBMCs derived from healthy donors as our T cell source. TCRs against tissue antigens such as PAP may be eliminated during negative selection in the thymus (50). T cells derived from in vitro cultures of non-thymocytes can function as a better source because these T cells do not undergo negative selection (51, 52). By searching for our defined PAP epitopes on T cells derived from these alternative sources, TCRs with higher affinity and specificity can be obtained.

[0087] The PAP-specific TCRs defined by the inventors can serve as a starting point for in vivo experiments and potential clinical development. The collected PAP epitope information can be used to prepare detection and capture reagents. It is recognized that future manipulations and improvements will be necessary to increase the potency of the candidate TCRs. Furthermore, the diversity of the inventors' candidate TCR pool can be increased by using T cells from alternative sources.

[0088] Table A: Exemplary Embodiments of the PAP TCR of the Present Invention Embodiments of the present invention include a composition of a substance comprising a polynucleotide encoding a TCR polynucleotide (e.g., a TCR polynucleotide arranged in a vector). In a typical embodiment, the polynucleotide encodes a Vα T cell receptor polypeptide and / or a Vβ T cell receptor polypeptide; the Vα / Vβ T cell receptor comprising the Vα T cell receptor polypeptide and / or Vβ T cell receptor polypeptide is CD8 + When expressed in T cells, CD8 + Vα / Vβ T cell receptors expressed by T cells are human leukocyte antigens (e.g., HLA-A * It is positioned within the vector to recognize / target the prostatic acid phosphatase peptide associated with 02:01).

[0089] Twenty-one exemplary functional TCR embodiments of the present invention are disclosed below.

[0090] 1. PAP-TCR-128 A. Target peptide sequence: LLLARAASLSL (SEQ ID NO: 59) ("PAP_A2_21"). Polypeptide sequence of the BV alpha-CDR3 region: CAASVDEKLTF (SEQ ID NO: 70) Polypeptide sequence of the CV beta-V region: CASSMYNEQFF (SEQ ID NO: 71) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0091] 2. PAP-TCR-131 A. Target peptide sequence: IMYSAHDTTV (SEQ ID NO: 55) ("PAP_A2_25"). Polypeptide sequence of the BV alpha-CDR3 region: CAVNANYGGATNKLIF (SEQ ID NO: 72) Polypeptide sequence of the CV beta-V region: CAISGGEVTTYEQYF (SEQ ID NO: 73) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0092] 3. PAP-TCR-137 A. Target peptide sequence: ILLWQPIPV (SEQ ID NO: 47) ("PAP_A2_14"). Polypeptide sequence of the BV alpha-CDR3 region: CATDAPTNFGNEKLTF (SEQ ID NO: 74) Polypeptide sequence of the CV beta-V region: CASSQRWTSGVWETQYF (SEQ ID NO: 75) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0093] 4. PAP-TCR-149 A. Target peptide sequence: ILLWQPIPV (SEQ ID NO: 47) ("PAP_A2_14"). Polypeptide sequence of the BV alpha-CDR3 region: CAASDNNDMRF (SEQ ID NO: 76) Polypeptide sequence of the CV beta-V region: CASSQTQGFGELFF (SEQ ID NO: 77) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0094] 5. PAP-TCR-154 A. Target peptide sequence: LLFFWLDRSVLA (SEQ ID NO: 62) ("PAP_A2_23"). Polypeptide sequence of the BV alpha-CDR3 region: CQGAQKLVF (SEQ ID NO: 78) Polypeptide sequence of the CV beta-V region: CASSGVGYETQYF (SEQ ID NO: 79) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0095] 6. PAP-TCR-156 A. Target peptide sequence: TLMSAMTNL (SEQ ID NO: 48) ("PAP_A2_22"). Polypeptide sequence of the BV alpha-CDR3 region: CAVNNARLMF (SEQ ID NO: 80) Polypeptide sequence of the CV beta-V region: CASSVAGSPEAFF (SEQ ID NO: 81) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka] [ka]

[0096] 7. PAP-TCR-168 A. Target peptide sequence: IRSTDVDRTL (SEQ ID NO: 52) ("PAP_A2_13"). Polypeptide sequence of the BV alpha-CDR3 region: CAASYPYTGRRALTF (SEQ ID NO: 82) Polypeptide sequence of the CV beta-V region: CAASYPYTGRRALTF (SEQ ID NO: 83) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0097] 8. PAP-TCR-173 A. Target peptide sequence: ILLWQPIPV (SEQ ID NO: 47) ("PAP_A2_14"). Polypeptide sequence of the BV alpha-CDR3 region: CAVEAYSGGYQKVTF (SEQ ID NO: 84) Polypeptide sequence of the CV beta-V region: CASSMYNEQFF (SEQ ID NO: 71) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0098] 9. PAP-TCR-175 A. Target peptide sequence: LLLARAASLSL (SEQ ID NO: 59) ("PAP_A2_21"). Polypeptide sequence of the BV alpha-CDR3 region: CAFEDSGYSTLTF (SEQ ID NO: 85) Polypeptide sequence of the CV beta-V region: CASGGLAGVDEQYF (SEQ ID NO: 86) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0099] 10. PAP-TCR-178 A. Target peptide sequence: LLLARAASLSL (SEQ ID NO: 59) ("PAP_A2_21"). Polypeptide sequence of the BV alpha-CDR3 region: CAASVDEKLTF (SEQ ID NO: 70) Polypeptide sequence of the CV beta-V region: CASSSYNEQFF (SEQ ID NO: 87) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0100] 11. PAP-TCR-204 A. Target peptide sequence: ILLWQPIPV (SEQ ID NO: 47) ("PAP_A2_14"). Polypeptide sequence of the BV alpha-CDR3 region: CAVGAGDYKLSF (SEQ ID NO: 88) Polypeptide sequence of the CV beta-V region: CASSQTTGQPQHF (SEQ ID NO: 89) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0101] 12. PAP-TCR-213 A. Target peptide sequence: IMYSAHDTTV (SEQ ID NO: 55) ("PAP_A2_25"). Polypeptide sequence of the BV alpha-CDR3 region: CAGAPETSGSRLTF (SEQ ID NO: 90) Polypeptide sequence of the CV beta-V region: CASSFGGGSSPLHF (SEQ ID NO: 91) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0102] 13. PAP-TCR-215-1 A. Target peptide sequence: LLLARAASLSL (SEQ ID NO: 59) ("PAP_A2_21"). Polypeptide sequence of the BV alpha-CDR3 region: CAASADEKLTF (SEQ ID NO: 92) Polypeptide sequence of the CV beta-V region: CASSQYNEQFF (SEQ ID NO: 93) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0103] 14. PAP-TCR-218 A. Target peptide sequence: LLLARAASLSL (SEQ ID NO: 59) ("PAP_A2_21"). Polypeptide sequence of the BV alpha-CDR3 region: CAASVDEKLTF (SEQ ID NO: 70) Polypeptide sequence of the CV beta-V region: CASSLYNEQFF (SEQ ID NO: 94) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0104] 15. PAP-TCR-219 A. Target peptide sequence: LLLARAASLSL (SEQ ID NO: 59) ("PAP_A2_21"). Polypeptide sequence of the BV alpha-CDR3 region: CAASADEKLTF (SEQ ID NO: 92) Polypeptide sequence of the CV beta-V region: CASSQYNEQFF (SEQ ID NO: 93) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0105] 16. PAP-TCR-220 A. Target peptide sequence: ILLWQPIPV (SEQ ID NO: 47) ("PAP_A2_14"). Polypeptide sequence of the BV alpha-CDR3 region: CAGRDNYGQNFVF (SEQ ID NO: 95) Polypeptide sequence of the CV beta-V region: CASSQVAGGTYEQYF (SEQ ID NO: 96) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka] [ka]

[0106] 17. PAP-TCR-223 A. Target peptide sequence: KVYDPLYCESV (SEQ ID NO: 58) ("PAP_A2_20"). Polypeptide sequence of the BV alpha-CDR3 region: CAVYGQNFVF (SEQ ID NO: 97) Polypeptide sequence of the CV beta-V region: CASSPIGLQETQYF (SEQ ID NO: 98) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0107] 18. PAP-TCR-224 A. Target peptide sequence: LLLARAASLSL (SEQ ID NO: 59) ("PAP_A2_21"). Polypeptide sequence of the BV alpha-CDR3 region: CAASEDEKLTF (SEQ ID NO: 99) Polypeptide sequence of the CV beta-V region: CASSLMAEQYF (SEQ ID NO: 100) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0108] 19. PAP-TCR-225 A. Target peptide sequence: LLLARAASLSL (SEQ ID NO: 59) ("PAP_A2_21"). Polypeptide sequence of the BV alpha-CDR3 region: CAASVDEKLTF (SEQ ID NO: 70) Polypeptide sequence of the CV beta-V region: CASSLQVEQFF (SEQ ID NO: 101) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0109] 20. PAP-TCR-226 A. Target peptide sequence: LLLARAASLSL (SEQ ID NO: 59) ("PAP_A2_21"). Polypeptide sequence of the BV alpha-CDR3 region: CAASADEKLTF (SEQ ID NO: 92) Polypeptide sequence of the CV beta-V region: CASSLFEEQYF (SEQ ID NO: 102) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0110] 21. PAP-TCR-228 A. Target peptide sequence: KVYDPLYCESV (SEQ ID NO: 58) ("PAP_A2_20"). Polypeptide sequence of the BV alpha-CDR3 region: CAGHLNARLMF (SEQ ID NO: 103) Polypeptide sequence of the CV beta-V region: CSAPRDGVYTF (SEQ ID NO: 104) D. Polynucleotide sequence of the alpha chain V(D)J region: [ka] E. Polynucleotide sequence of the beta chain V(D)J region: [ka]

[0111] Table B: Exemplary embodiments of the TCR variant of the present invention The following provides exemplary examples of variants of PAP-TCR-156. As noted in Table A above, these embodiments of the TCR of the present invention target a PAP peptide having the sequence:TLMSAMTNL (SEQ ID NO: 48).

[0112] 1.PAP-TCR-156-4(PAP-TCR-156-aCDR3-R7A) A. Polypeptide sequence of alpha-CDR1: DRGSQS (SEQ ID NO: 105) B. Polypeptide sequence of alpha-CDR2: IYSNGD (SEQ ID NO: 106) Polypeptide sequence of C. alpha-CDR3: CAVNNAALMF (SEQ ID NO: 107) Polypeptide sequence of D. beta-CDR1: SGDLS (SEQ ID NO: 108) Polypeptide sequence of E. betaCDR2: YYNGEE (SEQ ID NO: 109) Polypeptide sequence of F. beta-CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) Polynucleotide sequence of G.TCR alpha: [ka] Polynucleotide sequence of H.TCR beta: [ka]

[0113] 2.PAP-TCR-156-29(PAP-TCR-156-aCDR1-S4E) A. Polypeptide sequence of alpha-CDR1: DRGEQS (SEQ ID NO: 139) B. Polypeptide sequence of alpha-CDR2: IYSNGD (SEQ ID NO: 106) Polypeptide sequence of C. alpha-CDR3: CAVNNAALMF (SEQ ID NO: 107) Polypeptide sequence of D. beta-CDR1: SGDLS (SEQ ID NO: 108) Polypeptide sequence of E. betaCDR2: YYNGEE (SEQ ID NO: 109) Polypeptide sequence of F. beta-CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) Polynucleotide sequence of G.TCR alpha: [ka] Polynucleotide sequence of H.TCR beta: [ka] [ka]

[0114] 3.PAP-TCR-156-30(PAP-TCR-156-aCDR1-S6E) A. Polypeptide sequence of alpha-CDR1: DRGSQE (SEQ ID NO: 140) B. Polypeptide sequence of alpha-CDR2: IYSNGD (SEQ ID NO: 106) Polypeptide sequence of C. alpha-CDR3: CAVNNAALMF (SEQ ID NO: 107) Polypeptide sequence of D. beta-CDR1: SGDLS (SEQ ID NO: 108) Polypeptide sequence of E. betaCDR2: YYNGEE (SEQ ID NO: 109) Polypeptide sequence of F. beta-CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) Polynucleotide sequence of G.TCR alpha: [ka] Polynucleotide sequence of H.TCR beta: [ka]

[0115] 4.PAP-TCR-156-31(PAP-TCR-156-aCDR1-S6H) A. Polypeptide sequence of alpha-CDR1: DRGSQH (SEQ ID NO: 141) B. Polypeptide sequence of alpha-CDR2: IYSNGD (SEQ ID NO: 106) Polypeptide sequence of C. alpha-CDR3: CAVNNAALMF (SEQ ID NO: 107) Polypeptide sequence of D. beta-CDR1: SGDLS (SEQ ID NO: 108) Polypeptide sequence of E. betaCDR2: YYNGEE (SEQ ID NO: 109) Polypeptide sequence of F. beta-CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) Polynucleotide sequence of G.TCR alpha: [ka] Polynucleotide sequence of H.TCR beta: [ka]

[0116] 5. PAP-TCR-156-32 (PAP-TCR-156-aCDR1-S6N) A. Polypeptide sequence of alpha CDR1: DRGSQN (SEQ ID NO: 142) B. Polypeptide sequence of alpha CDR2: IYSNGD (SEQ ID NO: 106) C. Polypeptide sequence of alpha CDR3: CAVNNAALMF (SEQ ID NO: 107) D. Polypeptide sequence of beta CDR1: SGDLS (SEQ ID NO: 108) E. Polypeptide sequence of beta CDR2: YYNGEE (SEQ ID NO: 109) F. Polypeptide sequence of beta CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) G. Polynucleotide sequence of TCR alpha:

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[0117] 6. PAP-TCR-156-33 (PAP-TCR-156-aCDR2-N4H) A. Polypeptide sequence of alpha CDR1: DRGSQS (SEQ ID NO: 105) B. Polypeptide sequence of alpha CDR2: IYSHGD (SEQ ID NO: 143) C. Polypeptide sequence of alpha CDR3: CAVNNAALMF (SEQ ID NO: 107) D. Polypeptide sequence of beta CDR1: SGDLS (SEQ ID NO: 108) E. Polypeptide sequence of beta CDR2: YYNGEE (SEQ ID NO: 109) F. Polypeptide sequence of beta CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) Polynucleotide sequence of G.TCR alpha: [ka] Polynucleotide sequence of H.TCR beta: [ka]

[0118] 7.PAP-TCR-156-34(PAP-TCR-156-aCDR2-D6N) A. Polypeptide sequence of alpha-CDR1: DRGSQS (SEQ ID NO: 105) B. Polypeptide sequence of alpha-CDR2: IYSNGN (SEQ ID NO: 106) Polypeptide sequence of C. alpha-CDR3: CAVNNAALMF (SEQ ID NO: 107) Polypeptide sequence of D. beta-CDR1: SGDLS (SEQ ID NO: 108) Polypeptide sequence of E. betaCDR2: YYNGEE (SEQ ID NO: 109) Polypeptide sequence of F. beta-CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) Polynucleotide sequence of G.TCR alpha: [ka] Polynucleotide sequence of H.TCR beta: [ka]

[0119] 8.PAP-TCR-156-35(PAP-TCR-156-bCDR1-S1H) A. Polypeptide sequence of alpha-CDR1: DRGSQS (SEQ ID NO: 105) B. Polypeptide sequence of alpha-CDR2: IYSNGD (SEQ ID NO: 106) Polypeptide sequence of C. alpha-CDR3: CAVNNAALMF (SEQ ID NO: 107) D. Polypeptide sequence of beta CDR1: HGDLS (SEQ ID NO: 110) E. Polypeptide sequence of beta CDR2: YYNGEE (SEQ ID NO: 109) F. Polypeptide sequence of beta CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) G. Polynucleotide sequence of TCR alpha:

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[0120] 9. PAP-TCR-156-36 (PAP-TCR-156-bCDR1-S1N) A. Polypeptide sequence of alpha CDR1: DRGSQS (SEQ ID NO: 105) B. Polypeptide sequence of alpha CDR2: IYSNGD (SEQ ID NO: 106) C. Polypeptide sequence of alpha CDR3: CAVNNAALMF (SEQ ID NO: 107) D. Polypeptide sequence of beta CDR1: NGDLS (SEQ ID NO: 111) E. Polypeptide sequence of beta CDR2: YYNGEE (SEQ ID NO: 109) F. Polypeptide sequence of beta CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) G. Polynucleotide sequence of TCR alpha:

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[0121] 10.PAP-TCR-156-37(PAP-TCR-156-bCDR2-Y1H) A. Polypeptide sequence of alpha-CDR1: DRGSQS (SEQ ID NO: 105) B. Polypeptide sequence of alpha-CDR2: IYSNGD (SEQ ID NO: 106) Polypeptide sequence of C. alpha-CDR3: CAVNNAALMF (SEQ ID NO: 107) Polypeptide sequence of D. beta-CDR1: SGDLS (SEQ ID NO: 108) Polypeptide sequence of E. betaCDR2: HYNGEE (SEQ ID NO: 112) Polypeptide sequence of F. beta-CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) Polynucleotide sequence of G.TCR alpha: [ka] Polynucleotide sequence of H.TCR beta: [ka]

[0122] 11.PAP-TCR-156-38(PAP-TCR-156-bCDR2-N3H) A. Polypeptide sequence of alpha-CDR1: DRGSQS (SEQ ID NO: 105) B. Polypeptide sequence of alpha-CDR2: IYSNGD (SEQ ID NO: 106) Polypeptide sequence of C. alpha-CDR3: CAVNNAALMF (SEQ ID NO: 107) Polypeptide sequence of D. beta-CDR1: SGDLS (SEQ ID NO: 108) Polypeptide sequence of E. betaCDR2: YYHGEE (SEQ ID NO: 113) Polypeptide sequence of F. beta-CDR3: CASSVAGSPEAFF (SEQ ID NO: 81) Polynucleotide sequence of G.TCR alpha: [ka] [ka] Polynucleotide sequence of H.TCR beta: [ka]

[0123] 12.PAP-TCR-156-39(PAP-TCR-156-bCDR3-E10H) A. Polypeptide sequence of alpha-CDR1: DRGSQS (SEQ ID NO: 105) B. Polypeptide sequence of alpha-CDR2: IYSNGD (SEQ ID NO: 106) Polypeptide sequence of C. alpha-CDR3: CAVNNAALMF (SEQ ID NO: 107) Polypeptide sequence of D. beta-CDR1: SGDLS (SEQ ID NO: 108) Polypeptide sequence of E. betaCDR2: YYNGEE (SEQ ID NO: 109) Polypeptide sequence of F. beta-CDR3: CASSVAGSPHAFF (SEQ ID NO: 114) Polynucleotide sequence of G.TCR alpha: [ka] Polynucleotide sequence of H.TCR beta: [ka] [ka]

[0124] References: [ka] [ka] [ka] [ka] The present invention provides, for example, the following items: (Item 1) A composition comprising polynucleotides encoding a T cell receptor (TCR) alpha chain polypeptide and / or a TCR beta chain polypeptide, wherein the polynucleotides are arranged in a vector and the vector is CD8 + When transduced into T cells, the TCR alpha chain polypeptide and / or TCR beta chain polypeptide encoded by the polynucleotide recognize the polypeptide epitope of human prostatic acid phosphatase (PAP), CD8 + A composition that forms T cell receptors on T cells. (Item 2) The aforementioned T cell receptor is human leukocyte antigen HLA-A * Recognizes the polypeptide epitope of human prostatic acid phosphatase in combination with 02:01; and / or The composition according to item 1, wherein the T cell receptor recognizes a polypeptide epitope of human prostatic acid phosphatase selected from ILLWQPIPV (SEQ ID NO: 47), TLMSAMTNL (SEQ ID NO: 48), IRSTDVDRTL (SEQ ID NO: 52), IMYSAHDTTV (SEQ ID NO: 55), KVYDPLYCESV (SEQ ID NO: 58), LLLARAASLSL (SEQ ID NO: 59), and LLFFWLDRSVLA (SEQ ID NO: 62). (Item 3) The composition according to item 1, wherein the polynucleotide encodes an amino acid of the TCR variable region, and the vector comprises a vector polynucleotide encoding a TCR constant region fused in-frame with the TCR variable region. (Item 4) The composition according to item 3, wherein the polynucleotide is arranged in a cell. (Item 5) The aforementioned cells are human CD8 + The composition described in item 4, which is a T cell. (Item 6) The aforementioned CD8 +T cells were obtained from individuals diagnosed with cancer expressing human prostatic acid phosphatase antigen; heterologous TCRs were identified as CD8 + CD8 is expressed on the surface of T cells. + The composition according to item 5, wherein T cells are transduced with a vector containing a polynucleotide encoding a TCR Vα polypeptide combined with a polynucleotide encoding a TCR Vβ polypeptide, wherein the heterologous TCR recognizes a human prostatic acid phosphatase peptide associated with a human leukocyte antigen expressed on the surface of the cancer cells. (Item 7) The composition according to item 6, wherein the cancer is prostate cancer. (Item 8) The composition according to item 1, wherein the polynucleotide encodes at least 100 amino acid segments having at least 98% sequence identity with respect to the amino acids encoded by SEQ ID NOs: 1 to 42. (Item 9) The composition according to item 8, wherein the T cell receptor (TCR) alpha chain polypeptide and / or the TCR beta chain polypeptide encoded by the polynucleotide comprises amino acid substitution mutations. (Item 10) The composition according to item 8, wherein the polynucleotide encodes at least 10 amino acid segments encoded by SEQ ID NOs: 115 to 138. (Item 11) A method for inhibiting the growth of prostate cancer cells, The aforementioned prostate cancer cells were transduced with polynucleotides encoding T cell receptor (TCR) alpha chain polypeptide and TCR beta chain polypeptide, and CD8 + A step of combining with T cells, the CD8 +A method comprising the steps of: when transduced into and expressed in T cells, the alpha chain polypeptide and the TCR beta chain polypeptide can form T cell receptors that recognize polypeptide epitopes on human prostatic acid phosphatase (PAP) expressed on prostate cancer cells, thereby inhibiting the growth of the prostate cancer cells. (Item 12) The aforementioned T cell receptor is human leukocyte antigen HLA-A * Recognizes the polypeptide epitope of human prostatic acid phosphatase in combination with 02:01; and / or The method according to item 11, wherein the T cell receptor recognizes a polypeptide epitope of human prostatic acid phosphatase selected from ILLWQPIPV (SEQ ID NO: 47), TLMSAMTNL (SEQ ID NO: 48), IRSTDVDRTL (SEQ ID NO: 52), IMYSAHDTTV (SEQ ID NO: 55), KVYDPLYCESV (SEQ ID NO: 58), LLLARAASLSL (SEQ ID NO: 59), and LLFFWLDRSVLA (SEQ ID NO: 62). (Item 13) The aforementioned T cell receptor is human leukocyte antigen HLA-A * The method described in item 12 for recognizing the polypeptide epitope of human prostatic acid phosphatase in combination with 02:01. (Item 14) The method according to item 13, wherein the T cell receptor recognizes a polypeptide epitope of human prostatic acid phosphatase selected from ILLWQPIPV (SEQ ID NO: 47), TLMSAMTNL (SEQ ID NO: 48), IRSTDVDRTL (SEQ ID NO: 52), IMYSAHDTTV (SEQ ID NO: 55), KVYDPLYCESV (SEQ ID NO: 58), LLLARAASLSL (SEQ ID NO: 59), and LLFFWLDRSVLA (SEQ ID NO: 62). (Item 15) The method according to item 14, wherein the T cell receptor recognizes a polypeptide epitope of human prostatic acid phosphatase, including TLMSAMTNL (SEQ ID NO: 48). (Item 16) CD8 +The method described in item 11, which involves combining T cells in vivo to treat individuals with prostate cancer. (Item 17) The method according to item 12, wherein the polynucleotide encodes a segment of at least 100 amino acids having at least 98% sequence identity with respect to the amino acids encoded by SEQ ID NOs: 1 to 42. (Item 18) The method according to item 17, wherein the T cells express a T cell receptor comprising at least 10 amino acid segments encoded by SEQ ID NOs. 115 to 138. (Item 19) A method for evaluating a patient's immune response to prostate cancer or prostate cancer vaccination, the method comprising the step of observing the induction or activation of T cells obtained from a patient having prostate cancer or who has received prostate cancer vaccination, wherein The induction or activation of the aforementioned T cells was observed in response to T cell exposure to polypeptide epitopes present on human prostatic acid phosphatase (PAP). A method by which observed T cell induction or activation provides evidence of a patient's immune response to prostate cancer or prostate cancer vaccination. (Item 20) The method according to item 19, wherein the T cells express a T cell receptor that recognizes a polypeptide epitope of human prostatic acid phosphatase selected from ILLWQPIPV (SEQ ID NO: 47), TLMSAMTNL (SEQ ID NO: 48), IRSTDVDRTL (SEQ ID NO: 52), IMYSAHDTTV (SEQ ID NO: 55), KVYDPLYCESV (SEQ ID NO: 58), LLLARAASLSL (SEQ ID NO: 59), and LLFFWLDRSVLA (SEQ ID NO: 62).

Claims

[Claim 1] The invention described in the specification.

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