Human T cell receptor pairs reactive with HLA-A*02:01-restricted human prostatic acid phosphatase (PAP) epitopes.

Abstract

Aspects of the present disclosure relate to methods and compositions for preparing immune cells, including engineered T cells, with T cell receptors that target human prostatic acid phosphatase (PAP) and are useful in treating prostate cancer. In certain embodiments of the present invention, the T cell receptors that recognize / bind to PAP target 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 under 35 U.S.C. §119(e) of commonly assigned co-pending U.S. Provisional Patent Application No. 63 / 315,825, filed March 2, 2022, and entitled "HUMAN T CELL RECEPTOR PAIRS REACTIVE WITH HLA-A*02:01 RESTRICTED HUMAN PROSTATIC ACID PHOSPHATASE (PAP) EPITOPES," which is incorporated by reference herein.

[0002] Federal Funding Statement This invention was made with Government support under Grant No. 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 technology]

[0004] 2. 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 and the fourth leading cause of cancer-related deaths in men in developed countries worldwide.

[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 bones and lymph nodes. Prostate cancer can cause pain, difficulty urinating, erectile dysfunction, and other symptoms. Typically, prostate cancer develops most frequently in men over the age of 50, representing the most common patient group. However, prostate cancer remains undetected in most cases, even when it can be determined. Prostate cancer is typically determined by a physical exam or a screening blood test, such as a PSA (prostate specific antigen) test. If prostate cancer is suspected, the cancer is typically confirmed by removing a piece of the prostate (biopsy) and examining it under a microscope. Further tests, such as x-rays and bone scans, may be performed to determine whether the prostate cancer has spread.

[0006] The treatment of prostate cancer remains an unsolved problem. Conventional treatment methods such as surgery, radiation therapy, hormone therapy, chemotherapy (in some cases), proton beam therapy, or some combination of these can be applied to the treatment of prostate cancer. However, the age and underlying health of the man, as well as the extent of spread, appearance under a microscope, and the response of the cancer to early treatment are important in determining the outcome of the disease. Since prostate cancer is a disease that is typically diagnosed in older men, many patients will die of other causes before the slowly progressing prostate cancer can spread and cause symptoms. This makes the choice of treatment difficult. The decision of whether to treat localized prostate cancer (tumor contained within the prostate gland) with curative intent is a trade-off between the expected beneficial effects and adverse effects on the survival and quality of life of the patient.

[0007] As known in the art, the immune system plays an important role in the treatment and prevention of many diseases.According to the current state of knowledge, mammals provide various mechanisms to protect the organism, for example by identifying and killing tumor cells.For the purpose of the present invention, these tumor cells must be detected and differentiated from the normal (healthy) cells and tissues of the organism.

[0008] It is an object of the present invention to provide compositions and methods useful for the treatment of prostate cancer (PCa). Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention Prostatic acid phosphatase (PAP) is a well-known prostate / prostate cancer antigen that can serve as a target for cancer therapy. *The 02:01 restricted PAP epitope was defined using multiple physical methods (including weak acid elution, co-immunoprecipitation, and secreted MHC IP based on the ARTEMIS platform) coupled with liquid chromatography-mass spectrometry (LC-MS). The recovered PAP epitopes were then used to stimulate peripheral mononuclear cells (PBMCs) from more than 20 healthy donors. Reactive T cells isolated by the recently developed CLInt-seq and TCR alpha / beta sequencing technologies were analyzed by 10x Genomics single-cell TCR sequencing. Polynucleotides encoding the paired TCR alpha / beta chains were then introduced into normal human T cells and tested for their functionality. Using this methodology, we discovered 21 TCR alpha / beta polypeptide pairs that specifically recognize and are activated by seven distinct PAP peptides. All of these 21 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 cognate tetramers. These seven candidates can be successfully paired and stimulated in human T cells. At least one TCR (PAP-TCR-156) shows significant IFNγ signal 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 cognate TCR sequences are useful in novel cancer immunotherapy and vaccines.

[0010] The invention disclosed herein has several embodiments that take advantage of the above discoveries. Embodiments of the invention include, for example, compositions of matter comprising polynucleotides encoding T cell receptor (TCR) alpha chain polypeptides and / or TCR beta chain polypeptides; +When transduced and expressed in a T cell, the alpha chain polypeptide and / or the TCR beta chain polypeptide can form a T cell receptor that recognizes / binds to a polypeptide epitope on human prostatic acid phosphatase (PAP). In typical embodiments of the invention, the polynucleotide is placed in a vector that includes one or more regulatory sequences, etc., for expressing the polypeptide in a cell. Embodiments of the invention include the use of vectors transduced with such cells (e.g., CD8 + T cells).

[0011] In certain embodiments of the invention, the T cell receptor that recognizes / binds 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 invention, the T cell receptor that recognizes / binds PAP targets at least one polypeptide epitope selected from HLA-A * recognizes / binds to the 02:01 restricted epitope. In some embodiments of the invention, polynucleotides encoding T cell receptor (TCR) alpha or beta chain polypeptides (e.g., TCRs encoded by SEQ ID NO:1-SEQ ID NO:42) are engineered to introduce one or more mutations into a selected TCR polypeptide, typically to provide the TCR with greater susceptibility to target killing while retaining physiological affinity (e.g., catch-bond mutations). In certain embodiments of the invention, polynucleotides encoding T cell receptor (TCR) alpha or beta chain polypeptides encode a segment of at least 5 or at least 10 amino acids of the alpha V region and / or beta V region polypeptide sequences set forth in Table A and / or Table B.

[0012] Embodiments of the invention also include methods of inhibiting the growth of prostate cancer cells. Typically, these methods involve infecting prostate cancer cells with CD8 T cells transduced with polynucleotides encoding T cell receptor (TCR) alpha chain polypeptides and TCR beta chain polypeptides. + T cells; + When transduced and expressed in a T cell, the aforementioned alpha chain polypeptides and TCR beta chain polypeptides can form a T cell receptor that recognizes / binds to a polypeptide epitope on human prostatic acid phosphatase (PAP). In certain embodiments of the invention, the polynucleotide encoding the T cell receptor (TCR) alpha chain polypeptide or TCR beta chain polypeptide encodes an alpha CDR3 region polypeptide sequence and / or a beta V region polypeptide sequence as set forth in Table A; and / or the polynucleotide is as set forth in Table B. Typically, in these methods, a polynucleotide encoding a TCR alpha / beta polypeptide pair is transduced into a CD8 + T cells are transduced and then combined in vivo with prostate cancer cells to treat individuals suffering from prostate cancer.

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

[0014] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It should be understood, however, that the detailed description and specific examples have been given for the purpose of illustration and set forth certain embodiments of the present invention, but are not intended to limit the present invention. Many changes and modifications may be made within the scope of the present invention without departing from the spirit of the invention, and the present invention includes all such modifications. [Brief description of the drawings]

[0015] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1: Overall project flow chart and summary diagram of the TCR screening process. (Left) Both monoallelic and multiallelic HLA A0201 cell lines were processed by three different physical methods to extract peptides on MHC I. The sequences of these peptides were then identified by LC MS, yielding 27 distinct PAP epitopes. (Right) Peptides were then screened against PBMCs from over 20 individuals, and reactive clones were identified using the CLint seq protocol (see, e.g., utility: PCT Application No. PCT / US20 / 49055, incorporated herein by reference).

[0016] [Diagram 2] Figures 2A-2B: Schematic of and data from a T2 stabilization assay designed to assess peptide-MHC I stability. Figure 2(a) Schematic of the entire process of the T2 assay; Figure 2(b) Graphed data showing the slope of the natural logarithm of A2 fluorescence intensity versus diluted peptide concentration for various PAP peptides. The 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] [Diagram 3] Figure 3: Schematic and data from a study using a secreted form of MHC I single chain trimer to assess peptide-MHC stability. Figure 3(a) Diagram of SCT constructs; Figure 3(b) SDS-PAGE gel of the relative yield of each PAP SCT compared to the positive control (+) WT1 peptide RMFPNAPYL.

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

[0019] [Diagram 5] Figure 5: Data from functional testing of candidate TCRs observed using different methods. Figure 5(a) IFNγ results of different TCR constructs on PBMCs using peptide dilutions; F5: positive control for 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-engineered PBMCs and K562-A2; grey bars: TCR-engineered PBMCs and K562-A2-PAP. Figure 5(c) Cytotoxicity curves of TCR-156 in incucyte using total GFP signal of target cells to quantify target cell number; blue: TCR-156-engineered PBMCs with K562-A2 target cells; red: TCR-156-engineered PBMCs with K562-A2-PAP target cells.

[0020] [Figure 6-1]Figures 6A-6D: Data from functional testing of TCR variants observed using various methods. Figure 6A provides data from a study of variants of PAP-TCR-156 (see Table B), which show that the introduction of substitution mutations can enhance the potency of TCRs such as PAP-TCR-156 without loss of specificity. Figure 6B provides data from a study of PAP-TCR-156 variants killing K62 A2 cells without (left panel) and with (right panel) PAP, which show 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 a study showing PAP-TCR-156 variant-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). FIG. 6D provides data from a study of PAP-TCR-156 mutants showing that these embodiments of the invention exhibit cytotoxicity against PC3-A2-PAP at lower E:T ratios (4:1 ratio in left panel, 1:1 ratio in 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 functional enhancement in the PAP peptide titration assay. The left panel shows data from unmutated TCR156 (wt) and TCR156 variants 156-29, 156-30, 156-31, 156-32, 156-33, and 156-34; the right panel shows data from unmutated TCR156 (wt) and TCR156 variants 156-35, 156-36, 156-37, 156-38, and 156-39. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Invention In describing the embodiments, reference may be made to the accompanying drawings, which form a part of the invention and which are shown by way of illustration of specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention. Certain aspects of the 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 by reference.

[0023] Prostatic acid phosphatase (PAP) is a well-known prostate / prostate cancer antigen that can serve as a target for cancer therapy (Kantoff et al, NEJM, 2010 Jul 29;363(5):411-22). HLA-A *02:01 restricted PAP epitopes were defined using multiple physical methods (including weak acid elution (MAE), co-immunoprecipitation (CoIP), and secreted MHC IP (sMHC-IP) based on the ARTEMIS platform) coupled with liquid chromatography-mass spectrometry (LC-MS) (Figure 1). In total, 27 candidate PAP peptides were identified from 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 technologies were analyzed by 10x Genomics single-cell TCR sequencing (Figure 1) (Nesterenko et al, PNAS March 30, 2021 118(13)e2100106118). The paired TCR alpha / beta chains were then introduced into normal human T cells and tested for their functionality. From our previous findings (Figure 2; Tables 2, 3 in Mao et al.), we found 21 TCR alpha / beta pairs that are specifically recognized and activated by 7 distinct PAP peptides in the Jurkat-NFAT-GFP system. At least 7 of these 21 TCRs can be specifically stained by cognate tetramers. These 7 candidates can be successfully paired and stimulated in human T cells. This knowledge of PAP epitopes and cognate TCR sequences may be used to develop novel cancer immunotherapies and vaccines.

[0024] An embodiment of the present invention includes a composition of matter comprising one or more vectors comprising the TCR polynucleotides disclosed herein. A "vector" is a composition of matter comprising an isolated nucleic acid, which can be used to deliver said isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds (e.g., polylysine compounds and liposomes, etc.) that facilitate the transfer of nucleic acid into a cell. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors, etc.

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

[0026] Typically, the compositions of the invention comprise mammalian cells (e.g., CD8 +The subject cell includes a polynucleotide encoding a TCR Vα polypeptide in combination with a polynucleotide encoding a TCR Vβ polypeptide such that the subject cell can express a Vα / Vβ TCR on the surface of the subject T cell, where the Vα / Vβ TCR recognizes a PAP peptide associated with HLA. As used herein, the term "transduced" or "transfected" or "transformed" refers to a process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0027] In another aspect, the invention provides a method for administering one or more nucleic acids (e.g., nucleic acids disposed in a lentiviral vector) encoding a TCR as disclosed herein to T cells (e.g., CD8 T cells obtained from an individual diagnosed with cancer that express a PAP epitope recognized by the TCR). + The present invention also includes modified T cells having downregulated or knocked-out gene expression (e.g., modified T cells having an endogenous T cell receptor knocked out and an exogenous / introduced T cell receptor that recognizes the PAP peptide associated with HLA). As used herein, the term "knockdown" refers to the reduction of gene expression of one or more genes. As used herein, the term "knockout" refers to the elimination of gene expression of one or more genes.

[0028] The modified T cells described herein may be included in a composition for use in a therapeutic regimen. The composition may include a pharmaceutical composition and may further include a pharma- ceutically acceptable carrier. A therapeutically effective amount of a pharmaceutical composition comprising modified T cells may be administered. The pharmaceutical composition of the present invention may include modified T cells described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer (such as neutral buffered saline, phosphate buffered saline); carbohydrates (such as glucose, mannose, sucrose, or dextran, mannitol); proteins; polypeptides or amino acids (such as glycine); antioxidants; chelating agents (such as 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 bearing antigen-specific TCRs has therapeutic potential in the treatment of cancer. + Genetic engineering of T cells has the advantage of redirecting T cells to a selected antigen, such as a PAP epitope recognized by the TCR. In this context, in one embodiment, the present invention provides an effective amount of a modified CD8 + The present invention includes a method of stimulating a T cell-mediated immune response against a target cell or tissue in a subject, comprising administering T cells to the subject. In this embodiment, CD8 + T cells are modified as described elsewhere herein. Also, embodiments of the invention include multiple modified CD8 T cells that target multiple PAP epitopes. + For example, embodiments of the invention include administering at least two different modified CD8 T cells. + T cells (e.g., a second CD8 T cell that targets the PAP peptide associated with a second human leukocyte antigen) + The first modified CD8 targeting human leukocyte antigen-associated PAP peptide in combination with T cells + The method includes administering to the subject a first in vivo immune response to a cellular immune response (e.g., a cellular immune response

[0030] An embodiment of the present invention is a composition of matter comprising a polynucleotide encoding a T cell receptor (TCR) alpha chain polypeptide and / or a TCR beta chain polypeptide, said polynucleotide being placed in a vector, said vector being capable of inducing transcriptional activity of CD8 + The composition includes a T cell, wherein the alpha chain polypeptide and / or the TCR beta chain polypeptide encoded by said polynucleotide when transduced into a T cell forms a T cell receptor that recognizes a polypeptide epitope of human prostatic acid phosphatase (PAP). In certain embodiments of the invention, the T cell receptor is a human leukocyte antigen HLA-A * Recognizes a polypeptide epitope of human prostatic acid phosphatase in combination with 02:01. In some embodiments of the invention, 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). In certain of these embodiments, the polynucleotide encodes amino acids of a TCR variable region and the vector comprises a vector polynucleotide encoding a TCR constant region fused in-frame with the TCR variable region (see, e.g., U.S. Patent Application Publication Nos. 20220354889, 20200138865, 20210363245, and 20210155941; and Coren et al., Biotechniques. 2015 Mar 1;58(3):135-9 (which describes aspects of the MSGV Hu acceptor vector sold by addgene™)). Typically in these compositions, the polynucleotide is transfected into cells (e.g., human CD8 + Optionally, for example, the polynucleotide is placed into a CD8 T cell obtained from an individual diagnosed with a cancer (e.g., prostate cancer) that expresses the human prostatic acid phosphatase antigen. +Place in T cells; CD8 + T cells are differentiated into CD8 + The T cell is transduced with a vector comprising a polynucleotide encoding a TCR Vα polypeptide combined with a polynucleotide encoding a TCR Vβ polypeptide to be expressed on the surface of the T cell, where the heterologous TCR recognizes a human prostatic acid phosphatase peptide associated with a human leukocyte antigen expressed on the surface of the cancer cells.

[0031] In certain compositions of the 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 an alpha CDR1, alpha CDR2, alpha CDR3, beta CDR1, beta CDR2, or beta CDR3 polypeptide sequence) of a TCR polypeptide embodiment of the invention set forth in Table A or Table B below. In certain compositions of the invention, the polynucleotide encodes a segment of at least 100 amino acids having at least 98% sequence identity to the amino acids encoded by SEQ ID NO:1-SEQ ID NO:42. In some embodiments of the invention, the T cell receptor (TCR) alpha chain polypeptide and / or TCR beta chain polypeptide encoded by the polynucleotide comprises amino acid substitution mutations (e.g., SEQ ID NO:1-SEQ ID NO:42) of the wild-type TCR amino acid sequence selected to optimize its interaction with its cognate ligand (see, e.g., Sibener et al., Cell 174, 672-687, July 26, 2018; and Zhao et al., Science 376, 155 (2022), the contents of which are incorporated by reference herein). In exemplary examples of such variants, the polynucleotide encodes a segment of at least 5, 10, 25, 50, or 100 amino acids encoded by SEQ ID NO:115-SEQ ID NO:138.

[0032]

[0010] Embodiments of the invention include methods of killing cancer cells that express human prostatic acid phosphatase peptides associated with human leukocyte antigens expressed on the surface of the cancer cells. For example, embodiments of the invention include methods of killing prostate cancer cells by inducing CD8 + T cells, + The present invention also includes a method of inhibiting the growth of prostate cancer cells, comprising combining an alpha chain polypeptide and a TCR beta chain polypeptide, which when transduced and expressed in a T cell, 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 of these embodiments, the T cell receptor is a human leukocyte antigen HLA-A *Recognizing a polypeptide epitope of human prostatic acid phosphatase in combination with 02:01. In some embodiments of the invention, the T cell receptor is selected from the group consisting of 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 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, the polypeptide epitope of human prostatic acid phosphatase is selected from CD8 + The T cells are combined in vivo to treat individuals suffering from prostate cancer.Optionally, in these methods, the polynucleotide encodes a segment of at least 100 amino acids with at least 98% sequence identity to the amino acids encoded by SEQ ID NO:1 to SEQ ID NO:42 (as known in the art, sequence identity is the ratio of the number of identical amino acids over the aligned length between two aligned sequences / segments, expressed as a percentage).

[0033] The embodiments of the present invention include a method for evaluating a patient's immune response to prostate cancer or prostate cancer vaccination.Typically, these methods include observing the induction or activation of T cells obtained from a patient who has prostate cancer or has received a prostate cancer vaccination, where the induction or activation of T cells is observed in response to exposure of the T cells to a polypeptide epitope present on human prostatic acid phosphatase (PAP); the observed induction or activation of T cells provides evidence of the patient's immune response to prostate cancer or prostatic cancer vaccination.Optionally, in these methods, T cells express a T cell receptor that recognizes a polypeptide epitope of human prostatic acid phosphatase selected from the following: 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] The present invention includes a method for treating a disease or condition characterized by expression of PAP. The treatment methodology includes administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cells described herein. The term "subject" is intended to include a living organism (e.g., a mammal) capable of eliciting an immune response. A "subject" or "patient" as used herein may be a human or a non-human mammal. Non-human mammals include, for example, livestock and pet animals (mammals such as sheep, cows, pigs, dogs, cats, and mice). Preferably, the subject is a human. In a typical embodiment of the invention, the human has a cancer that expresses a PAP epitope recognized by the TCR. In some embodiments of the invention, the cells of the cancer form a solid tumor. In an exemplary embodiment of the invention, the cancer cells are prostate cancer cells.

[0035] Related embodiments of the invention include methods of preventing and / or treating an individual who has been diagnosed with, is suspected of having, or is at risk for developing or recurring with cancer, wherein said cancer comprises cancer cells expressing a PAP epitope recognized by a TCR. This approach involves administering to the individual modified human T cells comprising a recombinant polynucleotide encoding a TCR, wherein said T cells are capable of directly recognizing cancer cells expressing the PAP epitope recognized by the TCR, and wherein the direct recognition of the cancer cells comprises HLA class II restricted binding of the TCR to the PAP epitope recognized by the TCR expressed by the cancer cells.

[0036] The engineered CD8 + With respect to the use of T cells, the methods generally involve administering an effective amount (e.g., by intravenous or intraperitoneal injection) of CD8 + The method includes administering a composition comprising T cells to an individual in need thereof. Suitable pharmaceutical compositions may be adapted for administration by any suitable route, such as parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes. Such compositions may be prepared by any method known in the pharmaceutical art, such as by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0037] In another aspect, the invention relates to the use of a polynucleotide or modified CD8 as described herein in the manufacture of a medicament for the treatment of a disease or condition characterized by expression of PAP in a subject in need of such treatment. + In an exemplary embodiment of the invention, the disease is a cancer (e.g., prostate cancer) that expresses a PAP epitope disclosed herein.

[0038] The technology in this field has been developed appropriately, and some methods and materials known in the art can be adapted for use with the invention disclosed herein.Such methods and materials are disclosed, for example, in U.S. Patent Application Publication Nos. 20190247432, 20190119350, 20190002523, 20190002522, 20180371050, 20180057560, 20170029483, 20160024174, and 20150141347, the contents of which are incorporated 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, the contents of both of which are incorporated by reference herein. 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 "Publications disclosed herein, such as "antigen-specific TCR cloning." PNAS 2021;118(3)" are incorporated by reference to disclose and describe aspects, methods, and / or materials related to the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly used by those of ordinary skill in the art. Unless otherwise defined, all technical terms, notations, and other scientific terms or terminology used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and such definitions contained herein should not necessarily be construed as representing a substantial difference beyond common understanding in the art. EXAMPLES

[0040] Example 1: Physical and in silico immunopeptidome profiling of the cancer antigen prostatic acid phosphatase reveals a viable target for TCR isolation Certain aspects of the invention disclosed below are 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 by 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 (pMHC) and T cell receptors (TCRs). Uncovering the identity of peptides bound to MHC is crucial for discovering cognate TCRs and predicting potential toxicity. We performed a multimodal immunopeptidome analysis against prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including weak acid elution, co-immunoprecipitation, and secretory MHC precipitation, were used to identify HLA-A * We captured an exhaustive signature of PAP on 02:01. In total, 27 PAP peptides were identified, but only 5 of these peptides were predicted by the commonly used algorithm NetMHCpan4.0. Peripheral blood mononuclear cells (PBMCs) from over 20 healthy donors were screened with PAP peptides. 21 cognate TCRs against 7 distinct epitopes were identified using a single-cell isolation technique that detects intracellular IFNγ and TNFα. One TCR bound to full-length PAP and HLA-A *The results show reactivity against cell lines expressing both 02:01 and 02:02. Our results demonstrate that the combination of a multimodal immunopeptidome approach is productive in elucidating the target peptide and defining the originally 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 late-stage prostate cancer often undergo radical prostatectomy to remove the prostate gland (11). We selected prostatic acid phosphatase (PAP) among many 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. Secreted forms of PAP do not compete with TCR-PAP recognition because the interaction is restricted to peptides bound to MHC I.

[0043] Previous attempts to target PAP led to the first FDA-approved cancer vaccine, sipuleucel-T (Provenge) (15). Clinical trials showed a median improvement in overall survival of 4.1 months in men with metastatic castration-resistant prostate cancer (15). T cell proliferative responses in vaccinated individuals were measured by the stimulation index (SI) (15). SI is the ratio of T cell proliferation to T cell proliferation in T cells cultured with antigen. 3 A positive T cell proliferative response was defined as an SI of >5 measured 6 weeks after immunization (15). Among patients receiving Provenge, 27.2% of patients demonstrated a response to PAP compared with 8.0% in the placebo group (15). Also, a recent study showed that T cells from sipuleucel-T-treated patients were able to respond to PAP. +Visual evidence was provided that target cells could be lysed (16). Neither the presented PAP epitope nor the cognate TCR sequence have been defined at the molecular level. Recovery of TCRs that specifically recognize the PAP epitope could provide a product for potential therapeutic treatment.

[0044] In silico prediction of epitopes on MHC I is an important but insufficient strategy Several computational methods have been developed to predict peptide-MHC binding affinity using knowledge based on experimentally defined epitopes (17-19). In silico predictions can rapidly obtain results and are widely used (18). Previous attempts to identify PAP epitopes also relied mainly on motif-based predictions (20, 21). In a recent study, Wells et al. assembled a consortium (TESLA) that included 25 different platforms for comparison (22). Only 6% of predicted peptides were found to be immunogenic in pMHC multimer staining (22). Finding consensus among in silico pipelines is also difficult. The overlap between different algorithms in TESLA is limited: between any two randomly selected methods, only a median of 13% and a maximum of 62% were found (22). Defining peptides bound to MHC I by prediction alone is difficult for two reasons: 1. Selection of a single and best tool to use, and 2. Evaluation of false positives and false negatives of prediction results.

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

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

[0047] result: HLA-A * 02:01 Polymorphic immunopeptidome profiling of PAP Extensive HLA-A characterization of PAP was performed using both physical and in silico approaches. * 02:01 We defined immune peptidome signatures by applying the commonly used algorithm NetMHCpan4.0 to identify HLA-A * We profiled PAP epitopes on 02:01 (18). Forty PAP peptides were identified using the top 2 percentile as a cutoff and were then compared to HLA-A * 02:01 was selected as a potentially good binder (Supplementary Table 1 in Mao et al.).

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

[0049] Because 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 for MHC I released from the cell surface after lysis (24, 25, 33). Peptides bound to MHC I are then dissociated from the purified product and analyzed by LC-MS / MS. Twelve PAP peptides were recovered by CoIP (Supplementary Fig. 1a; Table 1 in Mao et al.). Two peptides overlapped with peptides found by MAE (Supplementary Fig. 1a; Table 1 in Mao et al.).

[0050] Secreted MHC-IP (sMHC-IP) was previously developed to enhance higher expression of an engineered soluble form of MHC I as a single-chain dimer (SCD) (26, 27). The recently published sMHC-IP platform, ARTEMIS, detects soluble HLA-A *We achieve robust expression and secretion of the 02:01 molecule (27). This engineered form contains a hexahistidine tag (6xHis tag) to increase the efficiency of enrichment with Ni-NTA agarose. Eight peptides were recovered in sMHC-IP, including six that were not found by the other two physical methods (Supplementary Fig. 1a in Mao et al., Table 1). Not a single peptide was found by all three methods (Supplementary Fig. 1a in Mao et al.; Table 1). Comparison between the physical and in silico approaches showed an overlap of only five peptides, which accounts for 12.5% ​​of the total peptides predicted by NetMHCpan4.0 (Supplementary Fig. 1b in Mao et al.; Table 1).

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

[0052] HLA-A of the recovered PAP peptides * 02:01 Specificity assessment Some of the peptides recovered by physical methods were non-HLA-A * The HLA-A of the recovered PAP epitopes may be derived from peptide fragments not present on the 02:01 subtype or MHC I. * To assess 02:01 specificity, a T2 cell binding assay was performed. The T2 cell line lacks the transporter associated with antigen processing (TAP) protein, which is responsible for loading of peptides onto MHC I. As a result, limited amounts of unstable MHC I (HLA-A * Only molecules containing 02:01 are naturally presented on T2 cells (35). For the T2 binding assay, chemically synthesized candidate peptides (>80% pure) are exogenously added to the growth medium.* Epitopes with 02:01 specificity are able to form stable peptide-MHC complexes and induce the accumulation of these molecules. * The amount of 02:01 can be quantified by anti-A2 antibody (clone BB7.2) conjugated with FITC using a flow cytometer (Figure 2a).

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

[0054] Peptide-MHC I complexes processed by endogenous mechanisms may show different stability compared to exogenous peptide pulsing (such as in T2 binding assays). This may be the result of post-translational modifications (PTMs). A recently developed technique, secreted single-chain trimers (SCT), was used to assess the relative stability of pMHC of interest (36). In the secreted SCT construct, the MHC I heavy chain (HLA-A with H74L and Y84C mutations) was expressed as a serovar I-peptide complex (SCT), whereas the exogenous peptide pulsing complex (SCT) was expressed as a serovar I-peptide complex (SCT). * The HLA-A alpha chain, light chain (beta-microglobulin), and corresponding peptide were tethered together as one single chain molecule by a linker (Figure 3a). The construct was expressed in cells and released into the culture supernatant. * Only peptides favored by 02:01 are expected to generate stable SCT and have higher yields. The amount of SCT was measured by band intensity in SDS-PAGE and was compared with that of HLA-A. *Normalization was performed against a known WT1 cancer epitope (RMFPNAPYL) restricted on 02:01 (Fig. 3b) (37, 38).

[0055] Nine peptides were associated with HLA-A when using a cutoff of 0.2 normalized to the control WT1 epitope. * 02:01 (Table 1 in Mao et al.; Fig. 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 positive in the T2 binding assay, suggesting that each evaluation technique may address different aspects of peptide-MHC stability (Table 1 in Mao et al.). Notably, PAP_A2_24 shows a higher yield (1.40) than the positive control, but a poor prediction score by NetMHCpan4.0 (24.22%) (Table 1 in Mao et al.; Fig. 3b).

[0056] Post-translationally modified PAP peptides bind to HLA-A * Increased binding affinity to 02:01 PAP-A2-24 inhibited HLA-A in different stability assays. *The results show contradictory results for 02:01 binding. One possible explanation is that PAP-A2-24 is post-translationally modified. Previous literature has reported N-glycosylation of PAP-A2-24 at asparagine (N220 of PAP) (39). To investigate whether an N-glycosylated form of PAP-A2-24 is presented, both PAP-A2-24 (SVHNFTLPSW (SEQ ID NO: 54)) and PAP-A2-25 (IMYSAHDTTV (SEQ ID NO: 55)) SCT products were treated with PNGaseF, which can specifically remove N-glycans (40). SDS-PAGE results show that PAP-A2-24 SCT showed a band with an apparent 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.). This result indicates that in addition to the glycosylation sites on the heavy and light chains of MHC I, additional N-glycans are present on PAP-A2-24. The only sequence differences between PAP-A2-24 SCT and PAP-A2-25 SCT are present in these peptide fragments. It is highly likely that additional N-glycans were present in the peptide (SVHNFTLPSW (SEQ ID NO: 54)).

[0057] The spectrum was also reanalyzed to confirm whether a deglycosylated form of PAP-A2-24 was also present. Previous reports have suggested that N-glycosylated asparagine (N) can be enzymatically deamidated to aspartic acid (D) (41). Both forms were detected by LC-MS in the CoIP results: SVHNFTLPSW (sequence number 54) and SVHDFTLPSW (sequence number 69) (Supplementary Figure 2b in Mao et al.).

[0058] Isolation of PAP peptide-specific TCRs from PBMCs of healthy individuals PBMC cells collected from multiple commercially available normal donors (n>20) were screened to find TCRs that were reactive to the PAP peptide. The 27 chemically synthesized peptides were added to total PBMCs, which contain a mixture of antigen-presenting cells (e.g., monocytes and B cells) that can prime T cells. T cells were then cultured and expanded in culture for 10 days. The CLint-seq protocol was then applied to stimulated cells to isolate reactive candidate T cells (30). As discussed above, TNFα + / IFNγ + Fixed CD8 T cell populations were sorted by FACS to enrich for reactive populations. TCR pairs that appeared more than once in the 10x Genomics sequencing results (frequency >1) were selected as potential PAP-reactive clones. 124 candidate α / β pairs were recovered from eight healthy individuals, including three females, four males, and one unknown (Supplementary Table 3 in Mao et al.).

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

[0060] The engineered TCR sequences were then cloned into a pMAX-cloning vector for rapid functional screening using electroporation. The pMAX constructs containing the TCR of interest were electroporated into the Jurkat-CD8-NFAT-GFP cell line used as a reporter system. In Jurkat-CD8-NFAT-GFP cells, GFP expression is induced by binding and activation of the NFAT promoter repeats following TCR activation (Figure 4a). GFP expression can then be quantified by flow cytometry to determine whether the TCR recognized the cognate peptide-MHC I. Mouse TCR beta chains were measured by FACS to estimate transfection efficiency. K562 cells were transfected with lentivirus to transfect HLA-A * 02:01-IRES-GFP transduced (K562-A2) were used as target cells in the study (Methods). Individual chemically synthesized PAP peptides were added to and presented by K562-A2 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] Validation of the function of candidate TCRs in human PBMCs Twenty-one candidate TCRs that showed reactivity in the Jurkat-CD8-NFAT-GFP system were then tested on human PBMC cells. Selected TCR constructs with mouse constant regions were tracked by truncated low affinity nerve growth factor receptor (delta LNGFR) as a transduction marker. Candidate TCRs were transduced into human PBMCs using the pMSGV retroviral system (9) (Methods). Surface dLNGFR levels were measured by FACS to estimate transduction efficiency. Mouse TCR beta chains were also quantified by FACS to evaluate whether the TCRs were transported to the cell surface. Tetramers containing individual PAP peptides of interest were produced and used on engineered PBMCs to ensure specific recognition (Supplementary Figure 3 in Mao et al.).

[0062] Stimulated T cells that recognize cognate peptides bound to MHC I can release cytokines such as IFNγ. ELISA was performed to quantify released IFNγ by using recombinant IFNγ as a standard (Methods). Individual PAP peptides were exogenously added to K562-A2 cells. Engineered PBMCs and target K562-A2 cells were mixed at a ratio of 2:1 (effector:target). Supernatants of the co-culture experiments were then harvested after 48 hours. Seven TCRs showed significant IFNγ signals against three distinct PAP peptides when expressed in human PBMCs (Figure 5a in Mao et al.; Table 1 in Mao et al.). Notably, five of these seven TCRs are against a peptide (PAP-21) that did not pass the prediction cutoff (<2%) using the NetMHCpan4.0 algorithm (Table 1 in Mao et al.).

[0063] TCRs showing high IFNγ signals in PBMCs were tested with serial dilutions of the cognate peptide to compare their relative potency with a clinically tested TCR, F5. This TCR against the MART1 epitope (EAAGIGILTV) was previously isolated from a melanoma patient (6). The F5 TCR is able to induce tumor regression in patients without affinity maturation to increase its potency and serves 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 at a 2:1 (effector:target) ratio. IFNγ ELISA was performed on supernatants collected after 48 hours. Notably, 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] PBMCs expressing these seven TCRs were then 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 by lentivirus into the K562-A2 cell line. The transduced population was single-cell sorted and expanded to generate clonal cell lines strongly expressing PAP. TCR-engineered PBMCs were mixed with target K562-A2-PAP cells at a ratio of 16:1 (effector:target). PBMCs transduced with F5 TCR and dLNGFR only (no TCR) empty vector were used as negative controls. ELISA was performed on co-culture supernatants after 48 hours. One TCR (PAP-TCR-156) showed specific full-length PAP recognition and produced 20,000 pg / ml IFNγ (Figure 5b). Four TCRs (128, 215-1, 218, and 219) produced low IFNγ signals (approximately 3,000 pg / ml) (FIG. 5b).

[0065] Cytotoxicity of candidate PAP TCRs was assessed by recording total target live cells using the IncuCyte platform. Target K562-A2-PAP cells co-express GFP and were imaged and analyzed in real time to detect GFP. - can be distinguished from PBMC cells. Live cell imaging was performed every 2 h to record the target cell number over a 120 h time course. The GFP signal was then processed by IncuCyte analysis tools to estimate the target cell area. One of the candidate TCRs, PAP-TCR-156, can inhibit the growth of cells expressing full-length PAP (Figure 5c). The total GFP area of ​​K562-A2-PAP is maintained at a similar level during 150 h of co-culture with PBMC expressing PAP-TCR-156 (Figure 5c). The total GFP area for K562-A2 cells showed a 3-fold increase as the negative control (Figure 5c).

[0066] Materials and Methods Weak acid elution: A weak acid elution protocol for eluting MHC I-associated peptides, based primarily on a previously published protocol with some modifications (53). 1–2 × 10 8cells were used. M202-PAP cells were dissociated with 1x PBS + 1mM EDTA, while K562-A2-PAP cells were harvested by spinning down at 1500RPM for 5 minutes. Target cells were then washed 3 times with 1x HBSS buffer (Thermo Fisher). 25ml of weak acid elution buffer (0.131M citric acid, 0.066M Na2HPO4, 150mM NaCl, 0.3uM aprotinin, 5mM iodoacetamide, pH 3.3) was applied to the target cells and gently shaken for 2 minutes at room temperature. Samples were then spun at 4000xg for 5 minutes at 4 degrees Celsius and the supernatant was collected. Formic acid was added to the samples to reach a final concentration of 0.1% (v / v). A 3ml 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 loaded onto the C18 column, followed by washing three times with 0.1% formic acid in water. The C18 column was then eluted three times with 200ul of 40% ACN + 5% formic acid + 55% H2O. The sample was then passed through a 3kd centrifugal filter (Millipore) at 4000xg for 90 minutes at 4 degrees Celsius. 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 M202-PAP cells or K562-A2 PAP cells were harvested either with non-enzymatic dissociation reagent (1x PBS + 1 mM EDTA) or by spinning down at 1500 rpm for 5 min. Cells were first washed 3 times with 1x PBS. CoIP lysis buffer (20 mM Tris (pH 8.0), 1 mM EDTA, 100 mM NaCl, 1% Triton® X-100, 60 mM n-octylglucoside, 1 mM PMSF (Sigma-Aldrich), protease inhibitors (Roche Life Science), and 1 mg / ml DNase I (Roche Life Science) was then added to 100 ml of lysate. 7Cells were lysed using 1 ml of lysis buffer per 10 cells. Samples were then shaken for 1 hour at 4° C. The lysate was then centrifuged at 10,000×g for 20 minutes to pellet debris. The supernatant was then transferred to 10 8 The lysate was combined with GammaBind Plus Sepharose beads (GE Lifesciences) conjugated with W6 / 32 antibody (BioXCell) at a ratio of 1 ml of beads per cell. The mixture of beads and lysate was shaken for 180 minutes at 4°C. The mixture was then loaded onto a Poly-Prep chromatography column (Bio-Rad). The column was then washed four times with 10 ml Wash Buffer I (CoIP Wash Buffer I: 20 mM Tris (pH 8.0), 1 mM EDTA, 100 mM NaCl, 60 mM n-octylglucoside, and 1 mg / ml DNase I), four times with 10 ml Wash Buffer II (CoIP Wash Buffer II: 10 mM Tris (pH 8.0)), and once with 10 ml ultrapure water (Thermo Fisher). Peptides were released from the beads by the addition of 10% acetic acid (Sigma) for 2 min and cleaned up by spinning at 3000×g for 30 s using 0.45 μm Costar Spin-X centrifuge filters (Corning). Samples were 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). Expression of secreted forms of both HLA-A2 and PAP was achieved by using a lentiviral transduction system in free style 293-F cells (Thermo Fisher). 400 ml of the supernatant containing secreted MHC I was purified by Ni-NTA agarose (1 μL slurry per ml supernatant). The slurry was loaded onto a Poly-Prep chromatography column and washed. The samples after denaturation were stored at -70 °C until further processing.

[0069] LC-MS analysis: The eluted samples were 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 samples were lyophilized by rapid vacuum and then reconstituted with water. The samples were then treated with a detergent removal kit to remove residual detergent from the lysis buffer. Finally, the samples were acidified to contain 5% formic acid before loading into the LC-MS. Samples were delivered to an Orbitrap Fusion Lumos hybrid mass spectrometer with a 140 min 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 data-dependent acquisition (DDA) mode: all MS scans were acquired under 120K resolution in an Orbitrap mass analyzer, singly and multiply charged ions above 800 m / z were selected to be fragmented using higher energy collision dissociation (HCD) at 32% collision energy, and then MS / MS scans were performed under 15K resolution in the Orbitrap. Dynamic exclusion allowed not to repeatedly select ions with the same m / z in a 60 s period. Data searching was performed using the Crux pipeline (v3.2) against the non-specifically digested EMBL human reference proteome (UP000005640human_9606), with PSM and peptide FDR set to a threshold of 1%.

[0070] T2 peptide binding assay: T2 cells (ATCC) were cultured in IMDM (Thermo Fisher) with 20% FBS (Omega Scientific). Prior to peptide loading, 2 × 10 5The cells were resuspended in 100ul of serum-free RPMI (Thermo Fisher) and added to each well of a 96U bottom tissue culture plate (Corning). Chemically synthesized peptides were diluted to multiple concentrations in serum-free RPMI and added to designated wells with T2 cells. Cells with peptides were co-cultured overnight at 37°C in an incubator. Cells were then washed twice with 1x PBS and stained with 2ul of anti-HLA-A2 FITC antibody (clone BB7.2, Biolegend) per well. The amount of HLA-A2 molecules was quantified by FACS.

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

[0072] Cell culture: K562 (ATCC), M202 (kindly provided by A. Ribas, UCLA), and Jurkat-NFAT-ZsGreen (kindly provided by D. Baltimore, Caltech) were cultured in RPMI1640 (Thermo Fisher) with 10% FBS (Omega Scientific) and glutamine (Fisher Scientific). 293T (ATCC) were cultured in DMEM (Thermo Fisher) with 10% FBS and glutamine. Naïve peripheral blood mononuclear cells (PBMCs) for stimulation were cultured in TCRPMI with 50U / ml IL-2 (Peprotech) and the PAP peptide of interest (purity >80%, Elim Biopharm) chemically synthesized as previously described (P Nesterenko, Cell Reports, 2021). TCR PMI 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 by 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: Isolation of reactive T cells by CLint-seq was performed on stimulated PBMCs according to a previously published protocol (30). After 7-10 days of co-culture with the PAP peptide pool, PBMCs were transferred to 96-well U-plates and left overnight. Cells were then cultured with 10ug / ml peptide pool and 1ug / ml CD28 / 49d antibody (BD Biosciences) for 1 hour, followed by the addition of Brefeldin A (Biolegend). After approximately 8 hours of incubation at 37°C, cells were processed as previously described and CD3+ T cells were identified by FACS. + / CD4 - / CD8 + / TNFα + / IFNγ + stained for populations ( 56 ).

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

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

[0076] Transduction of TCRs in PBMCs: Manipulation of candidate TCRs in PBMCs was performed according to a previous publication (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-engineered PBMCs at a ratio of 1:2 (T:E) in medium desired for the target cells and supplemented with 1 μg / ml anti-CD28 / CD49d antibody (BD Biosciences). For cell lines expressing full-length PAP, target cells were first treated with 2 ng / ml IFNγ and 3 ng / ml TNFα for 8–10 h. Target cells and PBMCs were then mixed at a ratio of 1:16 (T:E) for co-culture analysis. Supernatants were collected after 48 h and analyzed by ELISA (BD Biosciences) to estimate IFNγ concentrations.

[0079] Cytotoxicity analysis by IncuCyte: Target cells were plated on 96-well tissue culture plates coated with 0.001% poly-L-lysine (Sigma) and kept at 37° C. for approximately 2 hours. TCR-engineered PBMCs were then added to the desired wells at an effector:target ratio of 2:1 (peptide-pulsed target cells) or 16:1 (full-length PAP target cells). Plates with cell mixtures were analyzed by the IncuCyte system for 120 hours using GFP surface area to estimate T cell killing.

[0080] Consideration Our study, using a multiple immunopeptidome approach, identified 27 potential HLA-A * The 02:01 restricted PAP peptide is revealed. We were able to recover 21 candidate PAP TCRs against 7 of the defined epitopes. The 7 TCRs show reactivity against 3 distinct epitopes on peptide-pulsed target cells when engineered in human PBMC cells. Of these, one TCR (PAP-TCR-156) shows reactivity against full-length PAP and HLA-A * It is able to recognize peptides displayed on cell lines expressing the 02:01 allele.

[0081] All three physical assays (MAE, CoIP, and sMHC-IP) were able to generate immunogenic peptides. For TCRs that work efficiently in PBMCs, all three cognate 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 retrieving immunogenic epitopes.

[0082] Epitopes defined by physical methods can be used to develop reagents for PAP-specific T cells. The peptide of interest can be refolded into MHC-based multimers as detection and isolation reagents. The common form in which four peptide-MHC molecules are attached onto a streptavidin molecule is called a "tetramer". More complex versions of multimers were also available by adding additional fluorophores and increasing the number of MHC monomers (such as pentamers or dextramers) (43). The production of MHC multimers depends primarily on knowing the identity of the peptide. Seven of our PAP TCRs recovered against three distinct PAP peptides can be specifically stained by their cognate tetramers. Other candidates in our list can also be used in the creation of multimers. These reagents can be useful in pre-screening for PAP-reactive T cells in patients or healthy donors being treated with Provenge.

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

[0084] One of our candidate TCRs, PAP-TCR-156, shows the potential to recognize cell lines expressing full-length PAP. This TCR quality shows weak T cell responses by IFNγ and cytotoxicity assays. Increased potency of these candidate TCRs is required for future application and testing.

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

[0086] Also, by using alternative sources of T cells, TCRs with higher potency can be obtained. PBMCs from healthy donors were used as the source of our T cells. TCRs against tissue antigens such as PAP may have been eliminated during negative selection in the thymus (50). T cells from athymic in vitro cultures may serve as a better source, as these T cells are not subject to negative selection (51, 52). By searching for our defined PAP epitope on T cells from these alternative sources, TCRs with higher affinity and specificity can be obtained.

[0087] The PAP-specific TCRs we define can serve as a starting point for in vivo experiments and potential clinical development. The collected PAP epitope information can be used to create detection and capture reagents. It is recognized that future manipulations and improvements need to be carried out to increase the potency of candidate TCRs. Also, T cells from alternative sources can be used to increase the diversity of our candidate TCR pool.

[0088] Table A: Exemplary PAP TCR embodiments of the present invention

[0013] Embodiments of the invention include compositions of matter comprising a polynucleotide encoding a TCR polynucleotide (e.g., a TCR polynucleotide disposed in a vector). In exemplary embodiments, the polynucleotide encodes a Vα T cell receptor polypeptide and / or a Vβ T cell receptor polypeptide; + When expressed in T cells, CD8 + Vα / Vβ T cell receptors expressed by T cells bind human leukocyte antigens (e.g., HLA-A * The ribozyme is placed in the vector such that it recognizes / targets the prostatic acid phosphatase peptide associated with the ribozyme 3 (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 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 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 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 BV alpha CDR3 region: CAASDNNDMRF (SEQ ID NO:76) Polypeptide sequence of 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 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 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 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 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 BV alpha CDR3 region: CAFEDSGYSTLTF (SEQ ID NO:85) Polypeptide sequence of 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 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 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 BV alpha CDR3 region: CAGAPETSGSRLTF (SEQ ID NO:90) Polypeptide sequence of 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 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 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 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 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 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 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 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 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 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 mutant TCR embodiments of the invention The following provide illustrative examples of variants of PAP-TCR-156. As noted above in Table A, these TCR embodiments of the invention target the 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) 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: [ka] H. Polynucleotide sequence of 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) 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: [ka] H. Polynucleotide sequence of 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) 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: [ka] H. Polynucleotide sequence of 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) 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: [ka] H. Polynucleotide sequence of 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: [ka] H. Polynucleotide sequence of TCR beta: [ka]

[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) G. Polynucleotide sequence of TCR alpha: [ka] H. Polynucleotide sequence of 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) 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: [ka] H. Polynucleotide sequence of 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) C. Polypeptide sequence of alpha CDR3: CAVNNAALMF (SEQ ID NO: 107) D. Polypeptide sequence of beta CDR1: HGGLS (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: [ka] [ka] H. Polynucleotide sequence of TCR beta: [ka]

[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: [ka] H. Polynucleotide sequence of TCR beta: [ka]

[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) 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: HYNGEE (SEQ ID NO:112) F. Polypeptide sequence of beta CDR3: CASSVAGSPEAFF (SEQ ID NO:81) G. Polynucleotide sequence of TCR alpha: [ka] H. Polynucleotide sequence of 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) 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: YYHGEE (SEQ ID NO:113) F. Polypeptide sequence of beta CDR3: CASSVAGSPEAFF (SEQ ID NO:81) G. Polynucleotide sequence of TCR alpha: [ka] [ka] H. Polynucleotide sequence of 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) 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: CASSVAGSPHAFF (SEQ ID NO: 114) G. Polynucleotide sequence of TCR alpha: [ka] H. Polynucleotide sequence of TCR beta: [ka] [ka]

[0124] References: [ka] [ka] [ka] [ka]

Claims

1. 1. A composition of matter comprising a polynucleotide encoding a T cell receptor (TCR) alpha chain polypeptide and / or a TCR beta chain polypeptide, said polynucleotide being placed in a vector, said vector being capable of inducing transcriptional activity against CD8 ＋ When transduced into a T cell, the TCR alpha chain polypeptide and / or the TCR beta chain polypeptide encoded by the polynucleotide recognizes a polypeptide epitope of human prostatic acid phosphatase (PAP). ＋ A composition that forms a T cell receptor on a T cell.

2. The T cell receptor is a human leukocyte antigen (HLA-A). ＊ recognizing a polypeptide epitope of human prostatic acid phosphatase in combination with 02:01; and / or 2. The composition of claim 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).

3. The composition of claim 1 , wherein the polynucleotide encodes amino acids of a TCR variable region and the vector comprises a vector polynucleotide encoding a TCR constant region fused in-frame with the TCR variable region.

4. The composition of claim 3 , wherein the polynucleotide is placed in a cell.

5. The cells are human CD8 ＋ The composition of claim 4 which is a T cell.

6. The CD8 ＋ T cells are obtained from an individual diagnosed with a cancer that expresses the human prostatic acid phosphatase antigen; and a heterologous TCR is expressed by said CD8 ＋ The CD8 ＋ 6. The composition of claim 5, wherein a T cell is transduced with a vector comprising 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 cells of the cancer.

7. The composition of claim 6 , wherein the cancer is prostate cancer.

8. The composition of claim 1, wherein the polynucleotide encodes a segment of at least 100 amino acids having at least 98% sequence identity to the amino acids encoded by SEQ ID NO:1-SEQ ID NO:

42.

9. The composition of claim 8, wherein the T cell receptor (TCR) alpha chain polypeptide and / or the TCR beta chain polypeptide encoded by the polynucleotide comprises an amino acid substitution mutation.

10. The composition of claim 8, wherein the polynucleotide encodes a segment of at least 10 amino acids encoded by SEQ ID NO:115 to SEQ ID NO:

138.

11. 1. A method for inhibiting the growth of prostate cancer cells, comprising: The prostate cancer cells are subjected to transduction with a polynucleotide encoding a T cell receptor (TCR) alpha chain polypeptide and a TCR beta chain polypeptide. ＋ combining said CD8 T cells with ＋ when transduced and expressed in a T cell, said alpha chain polypeptide and said TCR beta chain polypeptide are capable of forming a T cell receptor that recognizes a polypeptide epitope on human prostatic acid phosphatase (PAP) expressed on said prostate cancer cells. thereby inhibiting growth of said prostate cancer cells.

12. The T cell receptor is a human leukocyte antigen (HLA-A). ＊ recognizing a polypeptide epitope of human prostatic acid phosphatase in combination with 02:01; and / or 12. The method of claim 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).

13. The T cell receptor is a human leukocyte antigen (HLA-A). ＊ 13. The method of claim 12, which recognizes a polypeptide epitope of human prostatic acid phosphatase in combination with 02:

01.

14. 14. The method of claim 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).

15. 15. The method of claim 14, wherein the T cell receptor recognizes a polypeptide epitope of human prostatic acid phosphatase comprising TLMSAMTNL (SEQ ID NO: 48).

16. CD8 ＋ 12. The method of claim 11, wherein the T cells are combined in vivo to treat an individual suffering from prostate cancer.

17. 13. The method of claim 12, wherein the polynucleotide encodes a segment of at least 100 amino acids having at least 98% sequence identity to the amino acids encoded by SEQ ID NO:1-SEQ ID NO:

42.

18. 18. The method of claim 17, wherein the T cell expresses a T cell receptor comprising a segment of at least 10 amino acids encoded by SEQ ID NO:115-SEQ ID NO:

138.

19. 1. A method for assessing a patient's immune response to prostate cancer or a prostate cancer vaccination, the method comprising observing the induction or activation of T cells obtained from a patient having prostate cancer or receiving a prostate cancer vaccination, wherein: the induction or activation of said T cells is observed in response to exposure of the T cells to a polypeptide epitope present on human prostatic acid phosphatase (PAP); The method, wherein the observed induction or activation of T cells provides evidence of a patient's immune response to prostate cancer or prostate cancer vaccination.

20. 20. The method of claim 19, wherein the T cell expresses 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).

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