A method for selectively amplifying cells expressing a mouse constant region TCR.

Abstract

Methods for selectively expanding the number of T cells are disclosed. The methods can include modifying human T cells to express a TCR, where the TCR comprises a mouse constant region; producing a cell population comprising some human T cells that express the TCR and some human T cells that do not express the TCR; and culturing the cell population in the presence of (i) irradiated feeder cells, (ii) one or more cytokines, and (iii) an antibody or antigen-binding portion thereof, where the antibody has antigen specificity for the mouse constant region of the TCR, to selectively expand the number of T cells that express the TCR relative to the number of T cells that do not express the TCR. Related cell populations, pharmaceutical compositions, and methods for treating or preventing cancer are also disclosed. [Selection diagram] None

Description

Technical Field

[0001] Cross - reference to Related Applications This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 568,339, filed Oct. 5, 2017, which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research and Development This invention was made with government support under Project No. Z1A BC 010985 by the National Institutes of Health, National Cancer Institute. The government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Material The computer - readable nucleotide / amino acid sequence listings, submitted herewith and identified as follows, are hereby incorporated by reference in their entirety: one 8,876 - byte ASCII (text) file named "740358_ST25.txt" dated Sep. 24, 2018.

Background Art

[0004] The treatment of cancer by administering cells modified to express an exogenous T - cell receptor (TCR) has produced constructive clinical results in some patients. Nevertheless, obstacles to the broader success of such therapies remain. For example, low delivery efficiency of the gene encoding the exogenous TCR may result in a low number of cells expressing the exogenous TCR. Thus, there is an unmet need for improved methods of manufacturing cells that express an exogenous TCR.

Summary of the Invention

[0005] Embodiments of the present invention provide a method for selectively amplifying the number of T cells, comprising: modifying human T cells to express a TCR, wherein the TCR includes a mouse constant region; producing a cell population comprising some human T cells expressing the TCR and some human T cells not expressing the TCR; and culturing the cell population in the presence of (i) irradiated feeder cells, (ii) one or more cytokines, and (iii) an antibody or its antigen-binding moiety, wherein the antibody has antigen specificity for the mouse constant region of the TCR, and selectively amplifying the number of T cells expressing the TCR relative to the number of T cells not expressing the TCR.

[0006] Further embodiments of the present invention provide a related cell population and a pharmaceutical composition comprising the cell population, which include a selectively amplified number of T cells prepared according to the method of the present invention.

[0007] A further embodiment of the present invention provides methods of treatment or prevention of cancer in mammals, the methods comprising selectively amplifying the number of T cells according to the methods of the present invention, and administering the selectively amplified number of T cells to a mammal in an amount effective for treating or preventing cancer in mammals. [Brief explanation of the drawing]

[0008] [Figure 1A] Figure 1A is a schematic diagram illustrating the nucleotide constructs encoding the human TCRα chain variable region (hVα), the mouse TCRα chain constant region (mCα), the synthetic linker sequence (L), the human TCRβ chain variable region (hVβ), and the mouse TCRβ chain constant region (mCβ). [Figure 1B] Figure 1B is a schematic diagram illustrating a method for selectively amplifying the number of mTCR-expressing T cells according to an embodiment of the present invention. [Figure 2]Figure 2 shows experimental data (dot plot) illustrating mTCRβ expression detected by FACS in cells electroporated with anti-mutant ERBB2 mTCR or anti-mutant ERBB2IP mTCR (ERBB2mutTCR or ERBB2IPmutTCR) after (i) CD3, CD4, or CD8 expression and (ii) selective amplification using the standard Rapid Expansion Protocol (REP) with OKT3 Ab or H57 Ab. T cells electroporated with electroporation buffer only (mock; without DNA / TCR) served as a negative control. The numbers in the dot plot represent the percentage of detected mTCR+ cells. [Figure 3A] Figures 3A and 3B are graphs showing the percentage (%) of anti-mutant ERBB2IP mTCRβ+ (ERBB2IPmutTCR) (A) or anti-mutant ERBB2 (ERBB2mutTCR) mTCRβ+ (B) cells expressing CD3, CD4, or CD8, detected after selective proliferation with various concentrations (ng / mL) of H57 Ab. T cells electroporated with electroporation buffer alone (mock; without DNA / TCR) served as a negative control. Electroporated cells that underwent standard REP with OKT3 Ab instead of selective proliferation with H57 Ab served as a positive control for nonspecific T cell proliferation. [Figure 3B] Figures 3A and 3B are graphs showing the percentage (%) of anti-mutant ERBB2IP mTCRβ+ (ERBB2IPmutTCR) (A) or anti-mutant ERBB2 (ERBB2mutTCR) mTCRβ+ (B) cells expressing CD3, CD4, or CD8, detected after selective proliferation with various concentrations (ng / mL) of H57 Ab. T cells electroporated with electroporation buffer alone (mock; without DNA / TCR) served as a negative control. Electroporated cells that underwent standard REP with OKT3 Ab instead of selective proliferation with H57 Ab served as a positive control for nonspecific T cell proliferation. [Figure 4A] Figure 4A is a schematic diagram illustrating a method for selectively amplifying the number of T cells expressing a mouse constant region (mTCR) including a TCR, according to an embodiment of the present invention. [Figure 4B] Figure 4B is a schematic diagram illustrating the binding of H57 Ab to the mouse TCRβ chain constant region (mCβ) of the TCR. Other components of the TCR include the human TCRα chain variable region (hVα), the mouse TCRα chain constant region (mCα), the synthetic linker sequence (L), and the human TCRβ chain variable region (hVβ). [Figure 5] Figures 5A-5D show experimental data (dot plots) illustrating CD3 expression and mouse TCRβ chain (mTCRβ) expression detected by FACS in untransfected, unstained cells (A), untransfected, stained cells (B), cells electroporated with electroporation buffer only (mock) (without TCR / transposon (Tn)) (C), and cells electroporated with the SBTS plasmid encoding mTCR (4149-TCRa2b2 / pSBSO) and the SBTS plasmid (pKan-CMV-SB11) encoding the transposase described in Example 3 (D). The numbers in the dot plots represent the percentage of cells with CD3+ / mTCRβ+ (upper right quadrant), CD3+ / mTCRβ- (lower right quadrant), CD3- / mTCRβ- (lower left quadrant), and CD3- / mTCRβ+ (upper left quadrant). [Figure 6] Figure 6 shows experimental data (dot plot) illustrating CD3 expression and mTCRβ expression detected by FACS in electroporated cells, after selective amplification of the initial number of electroporated cells (mTCR+ cells in REP mediated by H57 Ab at the indicated concentration (ng / mL)). Electroporated cells with only electroporation buffer (mock) (no TCR) served as negative controls. The numbers in the dot plot represent the percentage of CD3+ / mTCRβ+ (upper right quadrant), CD3+ / mTCRβ- (lower right quadrant), CD3- / mTCRβ- (lower left quadrant), and CD3- / mTCRβ+ (upper left quadrant) cells. [Figure 7] Figure 7 shows experimental data (dot plots) illustrating CD3 and mTCRβ expression detected by FACS in cells electroporated with mTCR, either stained or unstained, after a second amplification with H57 Ab or OKT3 Ab. Experimental data (dot plots) illustrating CD4 and CD8 expression detected by FACS in cells electroporated with mTCR after a second amplification with OKT3 Ab are also shown. [Figure 8] Figures 8A and 8B show experimental data (dot plots) illustrating CD8 expression and anti-MART-1 TCR expression detected by FACS in non-transduced (UT) cells (Figure 8B) and transduced cells (Figure 8A) before proliferation (pre-rapid amplification protocol (REP)). Figure 8C shows experimental data (dot plots) illustrating CD8 expression and anti-MART-1 TCR expression detected by FACS in transduced cells from Figure 8A before amplification (pre-REP) and after dilution to approximately 5% mTCRb+ cells. [Figure 9] Figure 9 shows experimental data (dot plots) illustrating CD8 expression and anti-MART-1 TCR expression detected by FACS in UT cells or diluted transduced cells from Figure 8C after amplification with (i) 50 CU or 500 CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"). Figures 3A and 3B are graphs showing the percentage (%) of anti-mutant ERBB2IP mTCRβ+ (ERBB2IPmutTCR) (A) or anti-mutant ERBB2 (ERBB2mutTCR) mTCRβ+ (B) cells expressing CD3, CD4, or CD8, detected after selective proliferation with various concentrations (ng / mL) of H57 Ab. T cells electroporated with electroporation buffer alone (mock; without DNA / TCR) served as a negative control. Electroporated cells that underwent standard REP with OKT3 Ab instead of selective proliferation with H57 Ab served as a positive control for nonspecific T cell proliferation. [Figure 10]Figure 10A is a graph showing the percentage of transduced cells detected in UT cells or diluted transduced cells from Figure 8C after amplification with (i) 50 CU or 500 CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"). Figure 10B is a graph showing the amplification factor achieved in UT cells or diluted transduced cells from Figure 8C after amplification with (i) 50 CU or 500 CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRB"). [Figure 11] Figures 11A-11C show experimental data (dot plots) illustrating CD8 expression and mTCRb expression detected by FACS before amplification of UT cells (Figure 11A) or cells transduced with anti-MART-1 TCR before (Figure 11B) or after (Figure 11C) 4-fold dilution. [Figure 12] Figures 12A-12E show experimental data (dot plots) illustrating CD8 expression and mTCRb expression detected by FACS after amplification of diluted TCR transducers from Figure 11C, either with UT cells (Figure 12E), or with OKT3 and 500CU IL-2 (Figure 12A), H57(mTCRb) and 500CU IL-2 (Figure 12B), H57(mTCRb) and 50CU IL-2 (Figure 12C), or H57(mTCRb) and no IL-2 (Figure 12D). [Figure 13] Figure 13A is a graph showing the percentage of transdextrins detected in UT cells or the diluted transdextrins shown in Figure 11C after amplification with (i) 0CU, 50CU, or 500CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"). Figure 13B is a graph showing the amplification factor achieved in the diluted transdextrins shown in Figure 11C after amplification with UT cells or the diluted transdextrins shown in Figure 11C after amplification with (i) 0CU, 50CU, or 500CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"). [Figure 14]Figures 14A to 14F show experimental data (dot plots) illustrating CD8 expression and mTCRb expression detected by FACS before amplification (Figure 14A) or after amplification of transduced cells with OKT3 (30 ng / ml) (Figure 14B), H57 (mTCRb) (5 ng / ml) (Figure 14F), 10 ng / ml (Figure 14E), 50 ng / ml (Figure 14D), or 500 ng / ml (Figure 14C)). [Figure 15] Figure 15 is a graph showing the amplification factor achieved for mTCR transduced cells after amplification with OKT3 (30 ng / ml) or H57 (mTCRb) (5, 10, 50, or 500 ng / ml). [Figure 16] Figures 16A-16E show experimental data (dot plots) illustrating CD8 expression and mTCRb expression detected by FACS after the second amplification of mTCR transduced cells that underwent the first amplification with H57. The second amplification was performed using OKT3 (30 ng / ml) (Figure 16A) or H57 (mTCRb) (5 ng / ml (Figure 16E), 10 ng / ml (Figure 16D), 50 ng / ml (Figure 16C), or 500 ng / ml (Figure 16B)). [Figure 17] Figure 17 is a graph showing the amplification factor achieved in mTCR transduced cells after the second amplification following the first proliferation with H57. The second amplification was performed with OKT3 (30 ng / ml) or H57 (mTCRb) (5, 10, 50, or 500 ng / ml). [Figure 18] Figure 18 shows experimental data (dot plot) illustrating CD3 expression and mTCRb expression detected by FACS the day after electroporation of PBMCs from donors 1 and 2 using 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2. Unstained PBMCs, untransfected PBMCs, and electroporated PBMCs with electroporation buffer only (mock) (no TCR) served as negative controls. [Figure 19]Figures 19A-19B show experimental data (dot plots) illustrating CD3 and mTCRb expression in PBMCs from donors 1 and 2 after transposition of 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2 and first amplification with H57 (Figure 19B). Unstained PBMCs (Figure 19A), electroporation buffer only (mock) (no TCR), and electroporated PBMCs (Figure 19B) served as negative controls. [Figure 20] Figures 20A-20B show experimental data (dot plots) illustrating CD3 and mTCRb expression in PBMCs from donors 1 and 2, which underwent transfer of 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2, followed by a first amplification with H57 and then amplification using standard REP (Figure 20B). Unstained PBMCs (Figure 20A), electroporation buffer only (mock) (no TCR), and electroporated PBMCs (Figure 20B) served as negative controls. [Figure 21]Figure 21A is a graph showing the concentration of IFN-γ secreted after co-culture of (i) DCs pulsed with DMSO, WT HUWE1 peptide, or mutant (mut) HUWE1 peptide, with (ii) cells from donor 1 to which 4149HUWE1-TCR1 had been transferred. DCs cultured alone served as a control. Mean ± SEM; n=3 technical replicates. Figure 21B is a graph showing the concentration of IFN-γ secreted after co-culture of (i) DCs pulsed with DMSO, WT TP53 peptide, or mut TP53 peptide, with (ii) cells from donor 1 to which 4149-TP53-TCRa2b2 had been transferred. DCs cultured alone served as a control. Mean ± SEM; n=3 technical replicates. Figure 21C is a graph showing the concentration of IFN-γ secreted after co-culture of (i) DCs pulsed with DMSO, WT HUWE1 peptide, or mut HUWE1 peptide with (ii) cells from donor 2 to which 4149-HUWE1-TCR1 had been transferred. DCs cultured alone served as a control. Mean ± SEM; n=3 technical replicates. Figure 21D is a graph showing the concentration of IFN-γ secreted after co-culture of (i) DCs pulsed with DMSO, WT TP53 peptide, or mut TP53 peptide with (ii) cells from donor 2 to which 4149-TP53-TCRa2b2 had been transferred. DCs cultured alone served as a control. Mean ± SEM; n=3 technical replicates. [Figure 22] Figure 22 is a schematic diagram illustrating a method for selectively amplifying the number of T cells expressing a mouse constant region (mTCR) including a TCR, according to an embodiment of the present invention. [Figure 23]Figure 23A is a graph showing the total number of cells in one cuvette measured at four time points during the method described in Example 14: after electroporation (white bars), after selective amplification with H57 (striped bars), after enrichment with H57-conjugated beads (gray bars), and after standard REP with OKT3 (black bars). Cells were electroporated with 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2. Triplicate cuvettes were advanced in parallel so that the data shown are mean + / - SEM (n=3). Figure 23B is a graph showing the percentage of CD3+ mTCR+ cells in one cuvette measured at four time points during the method described in Example 14: after electroporation (white bars), after selective amplification with H57 (striped bars), after enrichment with H57-conjugated beads (gray bars), and after standard REP with OKT3 (black bars). Cells were electroporated with either 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2. Triplicate cuvettes were advanced in parallel so that the data shown are mean + / - SEM (n=3). Figures 23C-23D are graphs showing the percentage of 4149-HUWE1-TCR1 metastatic (mTCR+) (Figure 23C) cells or 4149-TP53-TCRa2b2 metastatic (mTCR+) (Figure 23D) cells measured on day 28 (after OKT3 REP) using the method described in Example 14. PBMCs electroporated with electroporation buffer only (mock) (no TCR) served as a negative control. Mock cells are shown as (1), and TCR metastatic cells are shown as (2). Triplicate cuvettes were advanced in parallel so that the data shown are mean + / - SEM (n=3). Figure 23E is a graph showing the percentage of CD4+mTCRβ+ cells (white bars) or CD8+mTCRβ+ cells (black bars) measured on day 28 (after OKT3 REP) of the method described in Example 14. The TCRs were 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2. Triplicate cuvettes were advanced in parallel so that the data shown are mean + / - SEM (n=3). [Figure 24]Figure 24A is a graph showing the percentage of cells positive for the indicated marker, measured before (Day 1) (white bars) or after (Day 28) (black bars) cell number amplification. In the cells, 4149-HUWE1-TCR1 was translocated. Figure 24B is a graph showing the percentage of mTCRβ+ cells with the indicated phenotype, measured before (Day 1) (white bars) or after (Day 28) (black bars) cell number amplification. In the cells, 4149-HUWE1-TCR1 was translocated. The phenotypes are central memory T (TCM) cells, memory stem T cells (TSCM) cells, naive T cells (TN) cells, effector memory T cells (TEM) cells, and effector memory RA T cells (TEMRA) cells. Figure 24C is a graph showing the percentage of cells positive for the indicated marker, measured before (Day 1) (white bars) or after (Day 28) (black bars) cell number amplification. In the cells, 4149-TP53-TCRa2b2 was translocated. Figure 24D is a graph showing the percentage of mTCRβ+ cells with the indicated phenotype, measured before (Day 1) (white bars) or after (Day 28) (black bars) cell number amplification. In the cells, 4149-TP53-TCRa2b2 was translocated. [Figure 25] Figure 25A is a graph showing the concentration of IFN-γ secreted after co-culture of (i) WT HUWE1 peptide (white bar) or mut HUWE1 peptide (black bar) in DCs pulsed with DMSO or 10, 1, or 0.1 μg / mL with (ii) cells to which 4149-HUWE1-TCR1 had been transferred. Figure 25B is a graph showing the concentration of IFN-γ secreted after co-culture of (i) WT TP53 peptide (white bar) or mut TP53 peptide (black bar) in DCs pulsed with DMSO or 10, 1, or 0.1 μg / mL with (ii) cells to which 4149-TP53-TCRa2b2 had been transferred. [Modes for carrying out the invention]

[0009] Detailed description of the invention Embodiments of the present invention provide a method for selectively amplifying the number of T cells. The method may include modifying human T cells to express a TCR, the TCR comprising a mouse constant region (hereinafter, "mTCR"). The method of the present invention may provide one or more of a variety of advantages. For example, the method of the present invention may provide selective amplification of the number of T cells expressing an mTCR relative to the number of cells that do not express an mTCR. The method of the present invention may provide a cell population containing a higher proportion of cells expressing an mTCR compared to a cell population prepared by a method that does not selectively amplify the number of T cells as described herein. Although not bound by any particular theory or mechanism, it is believed that a cell population containing a higher proportion of cells expressing an mTCR may provide either or both improved destruction of target cancer cells and / or improved treatment of cancer compared to a cell population containing a lower proportion of cells expressing an mTCR.

[0010] A TCR generally comprises two polypeptides (i.e., polypeptide chains), such as the α-chain, β-chain, γ-chain, δ-chain, or a combination thereof. Such polypeptide chains of TCRs are known in the art. An mTCR may contain any amino acid sequence, provided that it includes a mouse constant region and can specifically bind to and immunologically recognize an antigen or its epitope, such as a disease-associated antigen.

[0011] mTCRs can be exogenous TCRs, i.e., TCRs that are not specific to T cells (do not occur naturally). Exogenous TCRs can be recombinant TCRs. Recombinant TCRs are TCRs produced by the recombinant expression of one or more genes encoding exogenous TCR α-, β-, γ-, and / or δ-chains. Methods for producing recombinant TCRs are known in the art.

[0012] In embodiments of the present invention, the mTCR comprises two polypeptide chains, each containing a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 of the TCR. Preferably, the mTCR comprises CDR1 of the α chain, CDR2 of the α chain, CDR3 of the α chain, CDR1 of the β chain, CDR2 of the β chain, and CDR3 of the β chain.

[0013] In one embodiment, the mTCR may include the amino acid sequence of the variable region of the TCR containing the above-mentioned CDR. In this regard, the TCR may include an α-chain variable region and a β-chain variable region.

[0014] In embodiments of the present invention, the mTCR further comprises a mouse constant region in addition to the variable region or CDR described above. Preferably, the mTCR comprises both an α-chain mouse constant region and a β-chain mouse constant region. As used herein, the term “mouse” means a mouse-derived TCR (or its components), i.e., a TCR (or its components) that originates from or has been expressed by mouse T cells, when referring to a TCR or any component of a TCR described herein (e.g., complementarity-determining region (CDR), variable region, constant region, alpha chain, and / or beta chain).

[0015] mTCR may include a TCRα chain containing a variable region and a constant region, and a TCRβ chain containing a variable region and a constant region. Hereinafter, the α-chain variable region and the β-chain variable region will be collectively referred to as the "variable region" of the TCR. Hereinafter, the α-chain constant region and the β-chain constant region will be collectively referred to as the "constant region" of the TCR.

[0016] In embodiments of the present invention, mTCR is a mouse TCR. A mouse TCR may comprise a polypeptide chain entirely derived from a mouse. In this regard, a mouse TCR may comprise a mouse variable region and a mouse constant region. Examples of mouse TCRs include, but are not limited to, those disclosed in U.S. Patent Nos. 8,216,565 and 9,487,573 and U.S. Patent Application No. 15 / 528,813.

[0017] In another embodiment of the present invention, the mTCR is a chimeric or hybrid TCR composed of amino acid sequences derived from TCRs from two different mammalian species, namely mouse and non-mouse species. For example, the mTCR may include a human variable region and a mouse constant region. Examples of chimeric TCRs including a human variable region and a mouse constant region are disclosed in Japanese Patent Application Nos. PCT / US2016 / 050875, PCT / US2017 / 044615, PCT / US2017 / 027865, U.S. Patent Publication Nos. 2013 / 0274203, 2017 / 0145070, and 2016 / 0152681; and U.S. Patent No. 8,785,601.

[0018] In embodiments of the present invention, the mTCR is an antigen-specific TCR. As used herein, the terms “antigen-specific” and “antigen-specific” mean that the mTCR specifically binds to an antigen or its epitope and is immunologically recognizable such that its binding to the antigen or its epitope triggers an immune response.

[0019] The antigen recognized by the antigen-specific mTCR can be any antigen characteristic of the disease. For example, the antigen may be, but is not limited to, a cancer antigen (also called a tumor antigen or tumor-associated antigen) or a viral antigen. Viral antigens are known in the art and include, for example, any viral protein, e.g., env, gag, pol, gp120, thymidine kinase, etc.

[0020] As used herein, the term “cancer antigen” refers to any molecule (e.g., protein, polypeptide, peptide, lipid, carbohydrate, etc.) that is expressed alone or primarily by tumor cells or cancer cells so that the antigen is associated with tumor or cancer. Cancer antigens may also be expressed by normal, non-tumor, or non-cancerous cells. However, in such cases, the expression of cancer antigens by normal, non-tumor, or non-cancerous cells is not as robust as that by tumor or cancer cells. In this regard, tumor cells or cancer cells may overexpress or express antigens at significantly higher levels compared to the expression of antigens by normal, non-tumor, or non-cancerous cells. Cancer antigens may also be expressed by cells in different stages of development or maturation. For example, cancer antigens may be further expressed by embryonic or fetal cells, which are not typically found in adult hosts. Alternatively, cancer antigens may be further expressed by stem cells or progenitor cells, which are not typically found in adult hosts. Cancer antigens are publicly known in the art, and examples include mesoserin, CD19, CD22, CD276 (B7H3), gp100, MART-1, epidermal growth factor receptor variant III (EGFRVIII), TRP-1, TRP-2, tyrosinase, mutant KRAS, NY-ESO-1 (also known as CAG-3), MAGE-1, and MAGE-3.

[0021] In one embodiment, the cancer antigen is a neoantigen. The neoantigen is a tumor-specific or cancer-specific antigen generated from a gene mutation occurring in tumor cells or cancer cells during neoplastic transformation. The neoantigen may be patient-specific. In a preferred embodiment, the neoantigen is an immunogenic neoantigen.

[0022] A cancer antigen may be an antigen expressed by any cell of any cancer or tumor, including the cancers and tumors described herein. A cancer antigen may be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor. Alternatively, a cancer antigen may be a cancer antigen of more than one type of cancer or tumor (e.g., characteristic of more than one type). For example, a cancer antigen may be expressed by both breast cancer cells and prostate cancer cells, and may not be expressed at all by normal, non-tumor, or non-cancerous cells.

[0023] A disease associated with or characterized by an antigen recognized by an antigen-specific mTCR can be any disease. For example, the condition may be cancerous or viral, as discussed herein.

[0024] Cancer can be any cancer, including any of the following: acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cancer, vulvar cancer, chronic lymphoblastic leukemia, chronic bone marrow cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharyngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, omentum and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer.

[0025] For the purposes of this specification, “viral disease” means a disease caused by a virus that can be transmitted from person to person or from organism to organism. In embodiments of the present invention, a viral disease is caused by a virus selected from the group consisting of herpesviruses, poxviruses, hepadnaviruses, papillomaviruses, adenoviruses, coronaviruses, orthomyxoviruses, paramyxoviruses, flaviviruses, and caliciviruses. For example, a viral disease may be caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, herpes simplex virus, Epstein-Barr virus, varicella virus, cytomegalovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV), human T lymphotropic virus, calicivirus, adenovirus, and arenavirus.

[0026] Viral diseases may include, for example, influenza, pneumonia, herpes, hepatitis, hepatitis A, hepatitis B, hepatitis C, chronic fatigue syndrome, idiopathic acute respiratory syndrome (SARS), gastroenteritis, enteritis, carditis, encephalitis, bronchiolitis, respiratory papillomatosis, meningitis, HIV / AIDS, and mononucleosis.

[0027] In embodiments of the present invention, the method comprises modifying human T cells to express mTCR. T cells may be isolated or purified. As used herein, the term “isolated” means taken out of its natural environment. As used herein, the term “purified” means having increased purity, and “purity” is a relative term and should not necessarily be interpreted as absolute purity. “Purified” T cells refer to T cells isolated from other natural components such as tissues, cells, proteins, nucleic acids, etc.

[0028] T cells can be any T cells, such as cultured T cells, e.g., primary T cells, or T cells derived from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from non-mouse mammals. When obtained from non-mouse mammals, T cells can be obtained from a multitude of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, spleen, or other tissues or body fluids. The cells may also be enriched or purified. Preferably, the T cells are human T cells. T cells are CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + This includes, but is not limited to, T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating cells, memory T cells, naive T cells, etc., and can be any type of T cell and any developmental stage. T cells are CD8 + These could be T cells or CD4+ T cells.

[0029] The method may include modifying human T cells to express mTCR using any suitable technique for introducing mTCR or nucleic acid encoding mTCR into human T cells, and obtaining mTCR expression by human T cells. Such techniques include, for example, Green and Sambrook. Molecular Cloning: A Laboratory ManualThis is described in the 4th edition, Cold Spring Harbour Press, Cold Spring Harbour, NY (2012). Examples of techniques that may be useful for modifying human T cells to express TCRs include, but are not limited to, transfection, transformation, transduction, electroporation, and gene editing techniques. In embodiments of the present invention, human T cells are modified using transposons, lentiviral vectors, or retroviral vectors. Examples of transposons include, but are not limited to, the SLEEPING BEAUTY transposon system (SBTS) (available from Intrexon (Germantown, MD) and Ziopharm (Boston, MA)), the PIGGYBAC transposon system (available from Transposagen, Lexington, KY), and the Tol2 transposon system. Examples of gene editing technologies include clustered regularly interspaced short palindromic repeats (CRISPR) / Cas systems (Cheng et al., Cell Res., 23:1163-71 (2013)), meganucleases, zinc finger nucleases (ZFNs), and transcriptional activation-like effector nucleases (TALENs).

[0030] The method may further involve producing a cell population containing some human T cells that express mTCRs and some human T cells that do not express mTCRs. Modifying human T cells to express mTCRs can be done with varying efficiencies. Therefore, modifying human T cells to express mTCRs may result in a mixed population of cells containing both mTCR-expressing and non-mTCR-expressing cells.

[0031] The method may further include culturing a cell population in the presence of (i) irradiated feeder cells, (ii) one or more cytokines, and (iii) an antibody or an antigen-binding portion thereof. Here, the antibody specifically binds to the murine constant region of the mTCR so as to selectively increase the number of T cells expressing mTCR relative to the number of T cells not expressing mTCR (also referred to herein as "selective amplification").

[0032] The irradiated feeder cells can include any irradiated feeder cells suitable for amplifying the number of T cells. In embodiments of the invention, the irradiated feeder cells include (i) irradiated allogeneic feeder cells; (ii) irradiated autologous feeder cells; or (ii) both (i) and (ii). The number of irradiated feeder cells used is not limited and can be selected by one of ordinary skill in the art depending on various factors such as, for example, the application and the desired number of cells obtained. For example, a multiple unit of irradiated feeder cells of 2×10 7 may be useful for amplification in small-scale studies. A multiple unit of irradiated feeder cells of 1×10 8 may be useful for medium-scale amplification. A multiple unit of irradiated feeder cells of about 2 to about 30 of 5×10 8 may be useful for large-scale amplification (e.g., clinical production) (usually up to about 1.5×10 10 ). For example, the method can use about 1×10 9 to about 4×10 9 allogeneic feeder cells and / or autologous feeder cells, preferably about 2×10 9 to about 3×10 9 allogeneic feeder cells and / or autologous feeder cells.

[0033] One or more cytokines can include any one or more cytokines suitable for amplifying the number of T cells. In embodiments of the invention, the one or more cytokines include any one or more of interleukin (IL)-2, IL-7, IL-12, IL-15, and IL-21.

[0034] The method may further include culturing a cell population in the presence of an antibody or its antigen-binding portion, wherein the antibody specifically binds to the mouse constant region of the mTCR. The antibody may be any type of immunoglobulin known in the art. For example, the antibody may be a recombinant antibody. The antibody may be any isotype, e.g., IgA, IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, etc. The antibody may be monoclonal or polyclonal. The antibody may be a naturally occurring antibody, e.g., an antibody isolated and / or purified from a mammal, e.g., a rabbit, goat, horse, chicken, hamster, human, etc. Alternatively, the antibody may be a genetically engineered antibody, e.g., a humanized antibody or a chimeric antibody. The antibody may be monomeric or multimeric. The antibody or its antigen-binding fragment may also have any level of affinity or avidity to the mouse constant region of the mTCR.

[0035] In embodiments of the present invention, the antibody comprises two polypeptide chains (a heavy chain and a light chain), each comprising a variable region containing antibody complementarity-determining regions (CDRs) 1, 2, and 3. The antibody may comprise heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3. In embodiments of the present invention, the antibody comprises a heavy chain variable region and a light chain variable region.

[0036] The antigen-binding portion of an antibody may be any portion having at least one antigen-binding site. In embodiments of the present invention, the antigen-binding portion may include the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody. In another embodiment of the present invention, the antigen-binding portion may include the heavy chain variable region and the light chain variable region of the antibody. The antigen-binding portion of an antibody may be a Fab fragment (Fab), an F(ab')2 fragment, a Fab' fragment, an Fv fragment, a single-stranded variable region fragment (scFv), a disulfide-stabilized variable region fragment (dsFv), scFv2CH3, scFv4, scFv3, scFv2, scFv-Fc, or (scFv)2. A single-stranded variable region fragment (scFv), which is a fusion protein containing the V domain of the antibody heavy chain linked to the variable (V) domain of the antibody light chain via a synthetic peptide, can be generated using conventional recombinant DNA techniques. Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology. However, the antigen-binding moieties of antibodies are not limited to these exemplary types. Antibodies and their antigen-binding moieties are collectively referred to as “antibodies” below unless otherwise specified.

[0037] The antibody may be any antibody that specifically binds to the mouse constant region of the mTCR. In embodiments of the present invention, the antibody specifically binds to the mouse constant region of the mTCR and does not bind to any portion of the human TCR, e.g., the human TCR constant region, or any portion of the human TCR complex. The TCR complex includes the α and β chains of the TCR and three dimeric signaling chains: CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. The CD3δ / ε and CD3γ / ε chains are collectively called the CD3 complex.

[0038] The antibody may specifically bind to the mouse constant region of the α-chain or the mouse constant region of the β-chain of the mTCR. In a preferred embodiment, the antibody specifically binds to the mouse constant region of the β-chain of the mTCR. The mouse constant region of the mTCRβ-chain to which the antibody specifically binds may include, or consist of, the amino acid sequence of SEQ ID NO: 1 (the amino acid sequence of the full-length mouse constant region of the mTCRβ-chain). The antibody may specifically bind to any portion of the mouse constant region of the mTCRβ-chain. For example, the antibody may specifically bind to the amino acid sequence of SEQ ID NO: 2 (the amino acid sequence of an exemplary minimal epitope in the full-length mouse constant region of the mTCRβ-chain). In embodiments of the present invention, the antibody specifically binds to amino acid residues D2, R4, N5, T7, E101, D103, K104, W105, P106, E107, G108, S109, and P110 of the amino acid sequence of SEQ ID NO: 1.

[0039] Antibodies that specifically bind to the mouse constant region of the mTCR are commercially available. For example, the H57 antibody (also known as H57-597) (available from Biolegend, San Diego, CA) specifically binds to the mouse constant region of the β chain of the mTCR. H57 is an IgG antibody from Armenian hamsters and is described, for example, in Kubo et al., J.Immunol., 142(8):2736-42 (1989) and Gregoire et al., PNAS, 88:8077-81 (1991). The epitope of the H57 antibody is described, for example, in Wang et al., EMBO J., 17(1):10-26 (1998). The H57 antibody specifically binds to amino acid residues D2, R4, N5, T7, E102, D104, K105, W106, P107, E108, G109, S110, and P111 of the amino acid sequence of SEQ ID NO: 1.

[0040] In embodiments of the present invention, the antibody includes a heavy chain variable region and a light chain variable region. For example, the antibody (Ab) may include, consist of, or substantially consist of the amino acid sequences of SEQ ID NO: 3 (variable region of the H57 Ab heavy chain) or SEQ ID NO: 4 (variable region of the H57 Ab light chain) or both SEQ ID NO: 3 and 4. Preferably, the antibody includes the amino acid sequences of both SEQ ID NO: 3 and 4. In embodiments of the present invention, the antibody includes complementarity-determining regions (CDRs) 1, CDR2, and CDR3 of the H57 Ab heavy chain of SEQ ID NO: 3, and CDRs 1, CDR2, and CDR3 of the H57 Ab light chain of SEQ ID NO: 4.

[0041] In embodiments of the present invention, the antibody comprises a heavy chain and a light chain containing the variable region described above. For example, the antibody may contain, consist of, or substantially consist of the amino acid sequences of SEQ ID NO: 5 (H57 Ab heavy chain) or SEQ ID NO: 6 (H57 Ab light chain), or both SEQ ID NO: 5 and 6. Preferably, the antibody contains both amino acid sequences of SEQ ID NO: 5 and 6.

[0042] As described herein, culturing a cell population in the presence of (i) irradiated feeder cells, (ii) one or more cytokines, and (iii) an antibody conveniently selectively amplifies the number of mTCR-expressing T cells relative to the number of non-mTCR-expressing T cells. In this regard, the method of the present invention can conveniently provide a cell population containing a higher proportion of mTCR-expressing cells compared to methods for amplifying cell numbers that do not use antibodies that specifically bind to the mouse constant region of the TCR. In embodiments of the present invention, the method increases the number of mTCR-expressing T cells from about 5 times or less to about 4,000 times or more. For example, the method of the present invention can increase the number of mTCR-expressing cells by approximately 5 to 4,000 times, 100 to 3,500 times, 1,000 to 3,000 times, 1,500 to 2,500 times, 10 to 1,000 times, 50 to 850 times, 100 to 900 times, 150 to 850 times, 200 to 800 times, 250 to 750 times, 300 to 700 times, 350 to 650 times, or 400 to 600 times. For example, the method of the present invention can increase the number of mTCR-expressing cells by about 10 times, about 50 times, about 100 times, about 150 times, about 200 times, about 250 times, about 300 times, about 350 times, about 400 times, about 450 times, about 500 times, about 550 times, about 600 times, about 650 times, about 700 times, about 750 times, about 800 times, about 850 times, about 900 times, about 950 times, about 1,000 times, or any two of the aforementioned values. The aforementioned amplification factors can be achieved over a period of about 10 to about 14 days, preferably about 14 days. The amplification factors achieved by the method of the present invention may be highly variable and donor-dependent.

[0043] In embodiments of the present invention, the method produces a selectively amplified cell population in which about 10% to about 95% of the cells express a TCR including a mouse constant region. In this regard, the method can produce a selectively amplified cell population in which about 10% to about 95%, about 15% to about 90%, about 20% to about 85%, about 25% to about 80%, about 30% to about 75%, about 35% to about 70%, or about 40% to about 65% of the cells express an mTCR. The method can produce a selectively amplified cell population in which approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or any two of the aforementioned values, express mTCR.

[0044] The method of the present invention can conveniently produce any number of mTCR-expressing T cells that may be suitable for any of the various applications. In embodiments of the present invention, the method produces approximately 1 × 10⁶ mTCR-expressing T cells. 6 ~Approx. 1×10 11 The above T cells can be produced. For example, the method of the present invention can produce approximately 1 × 10⁶ mTCR-expressing T cells in a small container (e.g., a T25 flask). 6 ~Approx. 1×10 7 The method of the present invention can produce approximately 5 × 10⁶ T cells expressing mTCR in a larger container (e.g., a T175 flask). 6 ~Approx. 3×10 7 The method of the present invention can produce approximately 1 × 10⁶ T cells expressing mTCR in another container (e.g., a GREX flask available from Wilson Wolf Manufacturing, New Brighton, MN). 9 ~Approx. 1×10 10 It is possible to produce T cells expressing mTCRs, approximately 1 × 10⁶ 6 ~Approx. 1×10 10 A population of T cells may be useful for small-scale screening experiments. A larger number of T cells expressing mTCRs, for example, about 1.5 × 10⁶ 10The cells described above can also be obtained, for example, for clinical application using the method of the present invention. The number of mTCR-expressing T cells produced by the method of the present invention is highly variable and may be donor-dependent.

[0045] The method may include performing one or fewer selective amplifications of the number of mTCR-expressing cells, or multiple selective amplifications of the number of mTCR-expressing cells. In embodiments of the present invention, the method includes performing one or more selective amplifications as described herein in relation to other aspects of the present invention, followed by one or more non-selective amplifications of the number of cells. In this regard, the method may further include culturing human T cells (also referred to herein as “non-selective amplification”) in the presence of (i) irradiated allogeneic feeder cells and / or irradiated autologous feeder cells, (ii) one or more cytokines, and (iii) an antibody that specifically binds to the human CD3 complex, or its antigen-binding moiety. Amplification of T cell numbers can be achieved by any of the many methods described, for example, in U.S. Patent No. 8,034,334; U.S. Patent No. 8,383,099; U.S. Patent Application Publication No. 2012 / 0244133; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). For example, T cell numbers can be amplified non-selectively using an antibody or its antigen-binding moiety that specifically binds to the human CD3 complex in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15) (IL-2 is preferred). An example of an antibody that specifically binds to the human CD3 complex is OKT3 (available from Ortho-McNeil, Raritan, NJ).

[0046] Multiple selective amplifications, or one or more non-selective amplifications following one or more selective amplifications, can increase the number of mTCRs by approximately 100,000 times or more. In embodiments of the present invention, the method produces a cell population in which approximately 20% to approximately 99% of the cells in the population express a TCR containing a mouse constant region. Multiple selective proliferations, or one or more non-selective proliferations following one or more selective proliferations, can produce a cell population in which approximately 25% to approximately 95%, approximately 30% to approximately 90%, approximately 35% to approximately 85%, approximately 40% to approximately 80%, approximately 45% to approximately 75%, or approximately 50% to approximately 70% of the cells in the population express mTCRs. The method can produce a cell population in which approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any two of the aforementioned values, of the cells express mTCR.

[0047] In embodiments of the present invention, the method further comprises separating T cells expressing a TCR from T cells not expressing a TCR using an antibody or its antigen-binding moiety that specifically binds to the mouse constant region of the TCR. In this regard, the method may include physically contacting a mixed population of cells, including cells expressing an mTCR and cells not expressing an mTCR, with the antibody so that the antibody specifically binds to the mouse constant region of the TCR. The antibody may be adhered to a support, e.g., beads. The method may further include washing the antibody and cells so that all or some of the cells not expressing an mTCR are removed from the cells expressing an mTCR. The method may further include eluting the mTCR-expressing cells from the antibody. Examples of techniques for separating T cells are described, for example, in Deniger et al., Mol. Ther., 24(6):1078-89 (2016) and Field et al., PLoS One, 8(6):e68201 (2013).

[0048] The methods of the present invention can conveniently provide a cell population enriched with cells expressing mTCR. Accordingly, embodiments of the present invention provide a cell population comprising some selectively amplified T cells prepared according to any of the methods described herein. The cell population may be a heterogeneous population comprising mTCR-expressing T cells together with at least one other cell (e.g., T cells that do not express mTCR) or non-T cell cells (e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc.). Alternatively, the cell population may be a substantially homogeneous population in which the population mainly comprises (e.g., substantially composed of) mTCR-expressing T cells. The population may also be a clonal population of cells in which all cells in the population are clones of a single mTCR-expressing T cell, so that all cells in the population express mTCR. In one embodiment of the present invention, the cell population is a clonal population comprising mTCR-expressing T cells.

[0049] The cell populations of the present invention can be isolated and / or purified. As used herein, the term “isolated” means taken out of the natural environment. As used herein, the term “purified” means increased purity, and “purity” is a relative term and should not necessarily be interpreted as absolute purity. Purity may be, for example, at least about 50%, about 60%, about 70%, about 80%, about 90%, over about 95%, or about 100%.

[0050] The cell populations of the present invention can be formulated into compositions such as pharmaceutical compositions. In this regard, the present invention provides a pharmaceutical composition comprising any of the cell populations described herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may contain any of the cell populations of the present invention in combination with other pharmaceutically active agents or drugs, such as chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and vincristine.

[0051] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to the pharmaceutical composition, the carrier may be one of those conventionally used for T cells. Methods for preparing administerable compositions are known or obvious to those skilled in the art and are described in detail, for example, in Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press (2012). A pharmaceutically acceptable carrier is preferably free from harmful side effects or toxicity under the conditions of use.

[0052] The choice of carrier may be determined by the specific method used to administer the cell population of the present invention. Accordingly, there are various suitable formulations of the pharmaceutical composition of the present invention. Suitable formulations may include those for parenteral, subcutaneous, intravenous, intramuscular, intra-arterial, intrathecal, intratumoral, or intraperitoneal administration. More than one route may be used to administer the cell population of the present invention, and in certain cases, one route may provide a more immediate and effective response than another.

[0053] Preferably, the cell population of the present invention is administered by injection, e.g., intravenously. pharmaceutically acceptable carriers for cells for injection may include, for example, ordinary saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or any isotonic carrier such as Ringer's lactate solution. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin. The pharmaceutical composition for injection may or may not contain IL-2. If the pharmaceutical composition contains IL-2, a concentration of about 300 IU / mL may be used.

[0054] For the purposes of the present invention, the amount or dose administered (e.g., the number of cells) should be sufficient to produce, for example, a therapeutic or prophylactic response in a mammal over a reasonable time frame. For example, the dose (e.g., the number of cells) should be sufficient to bind to a disease-related antigen or to detect, treat, or prevent a disease for a period of about 2 hours or more, e.g., about 12 to about 24 hours or more, from the time of administration. In certain embodiments, the period may be longer. The dose is determined by the efficacy of the cell population and the condition of the mammal (e.g., human), as well as the body weight of the mammal being treated (e.g., human).

[0055] Many assays for determining dosage are known in the art. For the purposes of the present invention, an assay may be used to determine the starting dose to administer to a mammal, comprising comparing the extent to which target cells are lysed or IFN-γ is secreted by mTCR-expressing T cells when a particular dose of T cells is administered to a pair of mammals, each given different doses of T cells. The extent to which target cells are lysed or IFN-γ is secreted when a particular dose is administered can be assayed by methods known in the art.

[0056] The dosage of the cell population of the present invention is also determined by the presence, nature, and extent of any adverse side effects that may be associated with the administration of a particular cell population. Typically, the attending physician determines the dosage of the cell population to treat each individual patient by considering various factors such as age, weight, general health, diet, sex, the cell population of the present invention to be administered, the route of administration, and the severity of the disease being treated. In one embodiment, the number of cells administered per infusion is, for example, about 1 × 10⁶ 6 Approximately 1 x 10⁻¹⁶ cells 12 It can vary by more than 1.5 × 10⁻¹⁶ units. In certain embodiments, at least about 1.5 × 10⁻¹⁶ units. 10 Cells may be administered.

[0057] Cell populations produced by the method of the present invention may be useful for treating or preventing diseases, such as cancer. Accordingly, another embodiment of the present invention provides a method for treating or preventing a disease in a mammal, the method comprising selectively amplifying a number of T cells according to any of the methods described herein with respect to other aspects of the present invention, and administering the selectively amplified number of T cells to a mammal in an amount effective for treating or preventing a disease in the mammal.

[0058] For the purposes of the method of the present invention, a population of cells is administered, which may be allogeneic or self-cells to the mammal. Preferably, the cells are self-cells to the mammal.

[0059] In embodiments of the present invention, the disease is cancer. The cancer may be any of the cancers described herein in relation to other embodiments of the present invention.

[0060] In embodiments of the present invention, the disease is a viral disease. The viral disease may be any of the viral diseases described herein in relation to other embodiments of the present invention.

[0061] As used herein, the term “mammal” refers to any mammal, including but not limited to rodents such as mice and hamsters, and mammals of the order Lagomorpha such as rabbits. It is preferable that the mammal is of the order Carnivora, which includes felines (cats) and canines (dogs). It is more preferable that the mammal is of the order Artiodactyla, which includes cats and pigs, or of the order Perissodactyla, which includes horses. It is most preferable that the mammal is of the order Primates, Ceboids, or Simoides (monkeys) or Homomorpha (humans and apes). Humans are a particularly preferred mammal.

[0062] As used herein, the terms “treatment” and “prevention,” and any words derived therefrom, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that a person skilled in the art would recognize as having a potential benefit or therapeutic effect. In this regard, the methods of the present invention may provide treatment or prevention of any amount or level of cancer in mammals. Furthermore, the treatment or prevention provided by the methods of the present invention may include the treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. For example, the treatment or prevention provided by the methods of the present invention may include promoting tumor regression. For the purposes of this specification, “prevention” may also include delaying the onset of the disease, or its symptoms or conditions.

[0063] The following embodiments further illustrate the present invention, but should not be interpreted as limiting its scope. [Examples]

[0064] Example 1 This embodiment demonstrates that selectively amplifying the number of T cells expressing a mouse constant region (mTCR) increases the number of cells expressing the mTCR.

[0065] RNA encoding two TCRs, one antigen-specific to the cancer-specific mutant ERBB2 neoantigen and the other to the cancer-specific mutant ERBB2IP neoantigen, was isolated from tumor-infiltrating lymphocytes (TILs) isolated from cancer patients. After amplifying cDNA from the RNA via reverse transcription, PCR amplification of the cDNA copies was performed. mTCR constructs (one per TCR) were prepared as described in Deniger et al., Mol. Ther., 24(6):1078-89 (2016). Briefly, the human TCR-α-VJ region was fused to the mouse TCR-α constant chain, and the human TCR-β-VDJ region was fused to the mouse TCR-β constant chain, with the synthetic linker sequence positioned between the alpha (α) and beta (β) chains (Figure 1A). Both mTCR constructs were synthesized and cloned into the SLEEPING BEAUTY Transposon System (SBTS) plasmid (available from Intrexon (Germantown, MD) and Ziopharm (Boston, MA)) (as described in Deniger et al., PLoS One, 10(6):e0128151 (2015) and Singh et al., Cancer Res., 71(10):3516-27 (2011)).

[0066] According to an embodiment of the present invention, peripheral blood mononuclear cells (PBMCs) are electroporated with mTCR, and mTCR + Figure 1B shows a schematic diagram illustrating a method for selectively amplifying the number of T cells. As shown in Figure 1B, using the AMAXA NUCLEOFECTOR II instrument and kit (Lonza, Basel, Switzerland), SBTS plasmids encoding one of each of two mTCRs (15 μg) and SBTS plasmids encoding a transposase (5 μg) are each applied to autologous PBMCs (2 × 10⁻¹⁶). 7 The cells were electroporated. After 24 hours, a mixed population of electroporated cells was obtained, containing cells expressing mTCR and cells not expressing mTCR.

[0067] As shown in Figure 1B, in a mixed population, T cells expressing the constant region of the mouse TCR (mTCR) + The number of mTCR cells was selectively amplified. + To selectively amplify T cells, a mixed population of electroporated cells (2 × 10⁻¹⁰ 6 Peripheral blood lymphocytes (PBLs) (2 × 10¹⁶) were irradiated with cells from (i) H57 antibody (Ab) (Biolegend, San Diego, CA) or OKT3 antibody, (ii) IL-2, and (iii) IL-21. 7 ) was co-cultured with it.

[0068] mTCR + After selectively amplifying the number of T cells, the cells were stained with (i) anti-CD3 Ab, anti-CD4 Ab, or anti-CD8 Ab and (ii) anti-mTCRβ antibody (H57). PBMCs electroporated with electroporation buffer alone (mock) (without TCR) served as a negative control.

[0069] Stained cells were counted by fluorescence-activated cell sorting (FACS). The results are shown in Figure 2. As shown in Figure 2, selective amplification with H57 Ab resulted in the identification of anti-mutant ERBB2 mTCR compared to OKT3. + T cell count and anti-mutant ERBB2IP mTCR + The number of T cells increased.

[0070] Example 2 This example shows that a higher concentration of H57 Ab leads to a greater number of mTCRs. + This demonstrates that it can be obtained.

[0071] As described in Example 1, PBMCs were electroporated with anti-ERBB2IP mTCR or anti-ERBB2 mTCR constructs. T cells expressing the constant region of mouse TCR in a mixed population (mTCR +The number of T cells was selectively amplified at various concentrations of H57 Ab (0 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 250 ng / mL, or 500 ng / mL) as described in Example 1.

[0072] After selective proliferation of mTCR+ T cells, cells were stained with (i) anti-CD3 Ab, anti-CD4 Ab, or anti-CD8 Ab and (ii) anti-mTCRβ antibody (H57). PBMCs electroporated with electroporation buffer alone (mock) (without TCR) served as a negative control. Cells electroporated after standard REP with OKT3 Ab (30 ng / mL) instead of selective proliferation with H57 Ab served as a positive control for nonspecific T cell proliferation.

[0073] The stained cells were counted by FACS. The results are shown in Figures 3A and 3B. As shown in Figures 3A and 3B, higher concentrations of H57 Ab resulted in a greater number of antimutant ERBB2 mTCR cells. + and anti-mutant ERBB2IP mTCR + We obtained cells.

[0074] Example 3 This embodiment demonstrates a method for selectively amplifying the number of T cells expressing mTCR.

[0075] RNA encoding a TCR with antigen specificity for the cancer-specific p53-Y220C neoantigen was isolated from tumor-infiltrating lymphocytes isolated from 4149 ovarian cancer patients. The cDNA was amplified from the RNA by reverse transcription, and then the cDNA copies were amplified by PCR. The mTCR construct was prepared by fusing the human TCR-α-VJ region to the mouse TCR-α constant chain, fusing the human TCR-β-VDJ region to the mouse TCR-β constant chain, and positioning the synthetic linker sequence between the α and β chains (Figure 4B). The mTCR construct was synthesized and cloned into an SBTS plasmid.

[0076] According to an embodiment of the present invention, PBMC is electroporated with mTCR, and mTCR + Figure 4A shows a schematic diagram illustrating a method for selectively amplifying the number of T cells. As shown in Figure 4A, autologous PBMCs were obtained from patients and cryopreserved. The cryopreserved PBMCs (200g) were thawed at 20-23°C for 10 minutes. The PBMCs were then placed in 50 / 50 medium (3 × 10⁶). 6 Cells were placed in medium (1 / mL) and incubated at 37°C for 2 hours. SBTS plasmids encoding mTCR and transposase were electroporated into autologous PBMCs using an AMAXA NUCLEOFECTOR II instrument and kit (Lonza, Basel, Switzerland). Human T cell NUCLEOFECTOR solution (300 μL) contained SBTS plasmid encoding mTCR (45 μg) and SBTS plasmid encoding transposase (15 μg). This human T cell NUCLEOFECTOR solution was added to cuvettes (100 μL / cubet). Electroporated cells were incubated overnight at 37°C in wells (5 mL 50 / 50 medium / well). After more than 18 hours, 50 U / mL benzonase was incubated at 37°C for 1 hour. A mixed population of electroporated cells, including cells expressing and not expressing mTCR, was obtained.

[0077] As shown in Figure 4A, after culturing at 37°C for more than 18 hours, T cells expressing the constant region of the mouse TCR (mTCR) in a mixed population were observed. + The number of mTCR cells was selectively amplified. + To selectively amplify T cells, mTCRs were induced in the presence of H57 Ab (Biolegend, San Diego, CA) (250 ng / mL), IL-2 (50 IU / mL), and IL-21 (30 ng / mL). + T cells (5×10 5 Peripheral blood lymphocytes (PBL) irradiated with radiation (1 x 10⁻¹⁰ 8 ) was co-cultured with it.

[0078] mTCR + After selective amplification of T cell numbers, mTCR +The number of T cells was further amplified using a standard rapid amplification protocol (REP) (see Example 5). In the standard REP, selectively amplified mTCR + T cells (5×10 6 ) irradiated PBL (5×10 8 The cells were co-cultured with OKT3 antibody (30 ng / mL) and IL-2 (3000 IU / mL). The OKT3 antibody reacts with an epitope in the epsilon subunit of the human CD3 complex.

[0079] Comparative Example 1 This example demonstrates the number of electroporated cells that express mTCR in the absence of selective amplification.

[0080] Autologous PBMCs were electroporated with SBTS plasmids encoding mTCR and transposase, as described in Example 4. The negative control consisted of untransfected autologous PBMCs, electroporation buffer only (mock), and electroporated PBMCs. The electroporated cells were stained with anti-CD3 antibody and anti-mTCRβ antibody (H57). Unstained, untransfected autologous PBMCs served as yet another negative control.

[0081] The day after electroporation, the stained cells were counted by FACS as described in Example 4, without selective amplification. FACS determined the lymphocyte / PI ratio. (neg) The cells were gated. PI is a stain for dead cells. PI (neg) Cells are living cells.

[0082] The results are shown in Figures 5A to 5D. As shown in Figures 5A to 5D, a small number of cells in the entire population (approximately 5% of all cells) expressed mTCR after electroporation without undergoing selective proliferation.

[0083] Example 4 This embodiment demonstrates that selective amplification of electroporated T cells with an H57 antibody selectively increases the number of cells expressing the mouse constant region (TCR) in a mixed population.

[0084] Autologous PBMCs were electroporated with SBTS plasmids encoding mTCRs and transposases, as described in Example 3. Various start numbers of mTCR+ T cells (1 × 10⁶) were then used. 5 , 5×10 5 , or 10 x 10 5 The ) was selectively amplified using various concentrations of H57 Ab (250 ng / mL, 500 ng / mL, or 750 ng / mL) as described in Example 3. In this experiment, the input was set to 5 × 10 based on the initial frequency. 5 The cells were standardized to be mTCR+ T cells. For example, 5% mTCR expression totaled 1 × 10⁶ 7 Transfer individual cells to the amplification protocol, 5 × 10 5 This meant achieving mTCR+ T cells. Electroporation buffer alone (mock) and electroporated PBMCs served as negative controls. Electroporated, selectively amplified cells were stained with anti-CD3 antibody and anti-mTCRβ antibody (H57).

[0085] Stained cells were counted by FACS. FACS was used to gate lymphocytes / PI(neg) cells. The results are shown in Figure 6 and Tables 1-2. As shown in Figure 6 and Tables 1-2, selective amplification with H57 Ab revealed the mTCR within the population. + The number of T cells increased.

[0086] [Table 1-1]

[0087] [Table 1-2]

[0088] [Table 2]

[0089] Example 5 In this embodiment, the first amplification by H57 Ab is followed by a second amplification by OKT3 Ab, which is mTCR + This demonstrates a further increase in the number of cells.

[0090] As described in Example 4, (i) initial number 5 × 10 5 mTCR+ T cells were subjected to (ii) selective proliferation with 250 ng / mL of H57 Ab (referred to as the "first amplification" in this example) and then electroporated. These cells were then further amplified in a GREX flask (Wilson Wolf Manufacturing, St. Paul, MN) using (i) standard REP with OKT3 Ab as described in Example 3, or (ii) H57 Ab as described in Example 4 (referred to as the "second amplification" in this example).

[0091] After the second amplification, the cells were stained with anti-CD3 antibody and anti-mTCRβ antibody (H57). The stained cells were counted by FACS. FACS was used to gate the cells to lymphocytes / PI(neg) cells. The results are shown in Figure 7.

[0092] As shown in Figure 7, the number of mTCR+ cells further increases with a second amplification using OKT3 Ab after the first amplification using H57 Ab. The number of mTCR+ cells increased significantly with the second amplification using OKT3 Ab compared to the second amplification using H57 Ab.

[0093] The percentage of viable cells, the total number of cells, the multiplier of change in the number of cells achieved by the second proliferation, the number of mTCR+ cells, and the multiplier of change in mTCR+ cells achieved by the second proliferation were also measured and compared in cells that underwent a second amplification with OKT3 Ab and H57 Ab. The results are shown in Table 3. As shown in Table 3, the mTCR was improved by the second amplification with OKT3 Ab after the first amplification with H57 Ab. + The number of cells increases further. Second amplification with OKT3 Ab compared to second amplification with H57 Ab, mTCR + The number of cells increased significantly.

[0094] [Table 3]

[0095] Example 6 This embodiment demonstrates that the number of cells expressing mTCR increases by selectively amplifying the number of T cells expressing mTCR.

[0096] PBLs were transduced on day 0 using a standard method with a gamma retroviral vector encoding mouse F5 (mF5) anti-MART-1 TCR. On day 10, the transduced cells were prepared for a second amplification. Amplification was performed using (i) various concentrations of IL-2, (ii) various ratios of feeder cells, and (iii) either OKT3 Ab (30 ng / ml) or H57 Ab (50 ng / ml) as described in Table 4.

[0097] [Table 4]

[0098] The FACS plots in Figures 8A-8B show the percentage of non-transduced cells and transduced cells expressing CD8 and mouse F5 (mF5) anti-MART-1 TCR (pre-rapid amplification protocol (REP)) before amplification with OKT3 Ab or H57 Ab. The transduction efficiency before amplification was 83.9%. This transduced population was diluted in cell culture medium to obtain approximately 5% mouse TCR beta-chain positive (mTCRb+) cells before amplification (Figure 8C).

[0099] Figures 9 and 10A-10B show the results of amplification of the cell number in the 5% mTCRb+ initiation population in Figure 8C, using (i) OKT3 (50 ng / ml) or H57 (50 ng / ml), and (ii) either 500 or 50 CU / IL-2. While the cell number was amplified approximately 1500-2300 times with OKT3, no selective amplification of the number of cells in the mTCRb+ cell population was observed. Alternatively, while the number of cells amplified by H57 was small (500-800 times), they showed a 4-8-fold increase in the percentage of mTCRb+ cells. Overall, the use of H57 in proliferation resulted in a greater number of mTCRb+ cells.

[0100] Example 7 This embodiment demonstrates that the number of cells expressing mTCR increases by selectively amplifying the number of T cells expressing mTCR.

[0101] PBL was transduced with a retroviral vector encoding mouse F5 TCR, as described in Example 6. The expression of CD8 and mTCRb in transduced cells (starting cell population) before amplification by H57 or OKT3 is shown in Figures 11A-11C. The starting mTCR+ population originally had 64.7% mTCRb+ cells (Figure 11B). The starting mTCR+ population was diluted fourfold to 14% mTCRb+ (Figure 11C).

[0102] The number of transduced cells and UT cells was amplified (i) without IL-2, with 50 CU IL-2, or with 500 CU IL-2, and (ii) using OKT3 Ab or H57 Ab. The results are shown in Figures 12A-12E and 13A-13B.

[0103] After amplification, the OKT3 population did not show selective amplification of the entire mTCRb+ cell population, which had nearly equal percentages (5%) of CD8+ and CD8- transduced cells. In comparison, both H57 amplification populations (500 CU / IL-2, respectively) showed selective mTCRb+ amplification of 31.2% and 59.1%, respectively. H57 amplification using 50 CU / IL-2 showed nearly twice as high amplification of mTCRb+ cells compared to cells grown in 500 CU / IL-2.

[0104] As shown in Figures 13-13B, the number of cells amplified by OKT3 or H57 was increased approximately 1400-1600 times in the presence of 500 CU / IL-2. However, with H57 amplification plus 50 CU / IL-2, the number was increased approximately 3500 times (more than twice that of the other conditions tested).

[0105] Example 8 This embodiment demonstrates that the number of cells expressing mTCR increases by selectively amplifying the number of T cells expressing mTCR.

[0106] PBL was transduced with a retroviral vector encoding mouse F5 TCR, as described in Example 6. The expression of CD8 and mTCRb in transduced cells (pre-REP) before amplification by H57 or OKT3 is shown in Figures 14A to 14F.

[0107] The number of transduced cells was amplified using (i) 50 CU IL-2 and (ii) OKT3 Ab (30 ng / ml) or H57 Ab (500, 50, 10, 5 ng / ml). The results are shown in Figures 14A-14F and 15. As shown in Figures 14A-14F and 15, the concentration of H57 antibody could reduce the titer by 10-fold without any adverse effect on selective mTCRb+ amplification. The overall amplification factor was similar across all groups tested. Preferential proliferation of CD8- cells after amplification with H57 was also observed.

[0108] Example 9 This example shows further amplification of the number of cells that underwent the first amplification by H57 in Example 8, through a second amplification by OKT3 or H57.

[0109] In Example 8, mTCR transduced cells amplified with H57 Ab were subjected to a second amplification with OKT3 or H57 to determine whether further enrichment of mTCRb+ cells could be achieved. The amplification conditions were the same as those described in Example 8.

[0110] The results are shown in Figures 16A-16E and Figure 17. The second amplification with OKT3 did not further enrich mTCRb+ cells, but the cell count increased by a further 1000-fold. The second amplification with H57 did not result in any further enrichment of mTCRb+ cells, although a slight decrease in mTCRb+ cells may have occurred at 500 and 50 ng / ml. The amplification factor in the population that underwent the second amplification with H57 was low, ranging from 200 to 500 times.

[0111] Example 10 This example demonstrates that mutation-specific TCRs are expressed by transposons the day after electroporation.

[0112] As shown in Figure 4A, autologous PBMCs were obtained from healthy donor 1 and healthy donor 2 and cryopreserved. The cryopreserved PBMCs were thawed and centrifuged at 20-23°C for 10 minutes (200g). The PBMCs were placed in 50 / 50 medium (3 × 10⁻¹⁶). 6 Cells were cultured at 37°C for 2 hours (cells / mL). SBTS plasmids encoding one of two mTCRs and an SBTS plasmid encoding a transposase were electroporated into autologous PBMCs using an AMAXA NUCLEOFECTOR II instrument and kit (Lonza, Basel, Switzerland). The two mTCRs were (1) 4149-HUWE1-TCR1 (class I) and (2) 4149-TP53-TCRa2b2 (class II). Human T cell NUCLEOFECTOR solution (300 μL) contained SBTS plasmids encoding the mTCRs (45 μg) and SBTS plasmids encoding the transposases (15 μg). After a 2-hour rest period, non-adherent PBMCs were collected by centrifugation (200 g) at 20-23°C for 10 minutes, the medium was removed, and the solution was replaced with human T cell NUCLEOFECTOR solution containing SBTS plasmids (100 μL added per cuvette). Electroporated cells were cultured overnight at 37°C in wells (5 mL 50 / 50 medium / well). After >18 hours, 50 U / mL benzonase was incubated at 37°C for 1 hour. A mixed population of electroporated cells was obtained, containing both cells expressing and not expressing mTCR.

[0113] The day after electroporation, electroporated cells were stained with (i) anti-CD3 Ab and (ii) anti-mTCRβ Ab. Unstained PBMCs, untransfected PBMCs, and electroporation buffer only (mock) (without TCR) and electroporated PBMCs served as negative controls. CD3 and mTCR expression in electroporated cells was measured by FACS (gate: lymphocytes / live cells (PI-)). The results are shown in Figure 18. As shown in Figure 18, mutation-specific TCRs were expressed by transposons the day after electroporation.

[0114] Example 11 This embodiment demonstrates that a single amplification by H57 results in a selective proliferation of mTCR+ T cells.

[0115] PBMCs from donors 1 and 2 were electroporated as described in Example 10. As shown in Figure 4A, after culturing at 37°C for more than 18 hours, T cells expressing the constant region of the mouse TCR (mTCR) in the mixed population were electroporated. + The number of mTCR cells was selectively amplified. + To selectively amplify the number of T cells, mTCR + T cells (5×10 5 ) irradiated PBL (1×10 8 The cells were co-cultured in the presence of H57 Ab (Maine Biotechnology Services, Portland, ME) (250 ng / mL), IL-2 (50 IU / mL), and IL-21 (30 ng / mL).

[0116] Selectively amplified cells were stained and evaluated by FACS as described in Example 10. Unstained PBMCs (Figure 19A), electroporation buffer only (mock) (without TCR), and electroporated PBMCs (Figure 19B) served as negative controls. The results are shown in Figures 19A-19B. In Figures 19A-19B, the cell count was determined on day 14 after one amplification cycle with H57 from a single T175 flask. As shown in Figures 19A-19B, a single amplification cycle with H57 resulted in selective proliferation of mTCR+ T cells.

[0117] Example 12 This embodiment demonstrates that further amplification of the number of cells that have undergone a first proliferation with H57, accompanied by a second proliferation with OKT3, in a gas-permeable flask, results in a significant amplification of the number of TCR-transferable T cells.

[0118] PBMCs from donors 1 and 2 were electroporated as described in Example 10. The number of metastatic cells was selectively increased using the H57 antibody as described in Example 11. mTCR + After selective amplification of T cell numbers, mTCR + The number of T cells was further amplified using standard REP. In standard REP, selectively amplified mTCR + T cells (5×10 6 ) irradiated PBL (5×10 8 ), OKT3 antibody (30 ng / mL), and IL-2 (3000 IU / mL) were co-cultured.

[0119] Selectively amplified cells were evaluated by staining and FACS as described in Example 10. Unstained PBMCs (Figure 20A), electroporation buffer only (mock) (no TCR), and electroporated PBMCs (Figure 20B) served as negative controls. Table 5 shows the number of cells 14 days after standard REP from one GREX-100 gas-permeable flask (Wilson Wolf Corporation, St. Paul, MN). Table 6 shows the percentage of mTCR+ cells 14 days after selective amplification with H57.

[0120] [Table 5]

[0121] [Table 6]

[0122] The results are shown in Figures 20A-20B. As shown in Figures 20A-20B, a significant increase in the number of TCR-transferred T cells was achieved by further amplification of the number of cells that had undergone the first proliferation by H57, accompanied by a second amplification by OKT3 in a gas-permeable flask.

[0123] Example 13 This embodiment demonstrates that TCR-transferred T cells selectively amplified with H57 antibody are specific to the allomutated neoantigen.

[0124] PBMCs from donors 1 and 2 were electroporated as described in Example 10. The number of electroporated cells was selectively amplified using the H57 antibody as described in Example 11. 4149-HUWE1-TCR1 recognizes mutant HUWE1. 4149-TP53-TCRa2b2 recognizes mutant TP53(Y220C).

[0125] Immature DCs were pulsed with wild-type (WT) HUWE1 peptide, mutant HUWE1 peptide, WT TP53, or mutant TP53. HUWE1 peptide was pulsed at 1 μg / mL. TP53 peptide was pulsed at 10 ng / mL. 5 × 10 4 Individual peptide pulse immature DCs and 10 5 Co-cultures of individual T cells were incubated overnight at 37°C. DCs were cultured individually, and DMSO-pulsed DCs served as controls. IFN-γ secretion was measured by ELISA (enzyme-linked immunosorbent assay).

[0126] The results are shown in Figures 21A to 21D. As shown in Figures 21A to 21D, TCR-transferred T cells selectively amplified with the H57 antibody recognized the allomutated neoantigen.

[0127] Example 14 This embodiment demonstrates a method for selectively amplifying the number of T cells expressing mTCR.

[0128] PBMC is electroporated with mTCR, and mTCR is processed according to the embodiments of the present invention. +Figure 22 shows a schematic diagram illustrating a method for selectively amplifying the number of T cells. As shown in Figure 22, autologous PBMCs were obtained from patients and cryopreserved. Some PBMCs were depleted of CD4+ cells using an LD column. The cryopreserved PBMCs were thawed in complete culture medium (CM) and centrifuged at 175g at approximately 22°C for 10 minutes. The cells were washed in Hanks equilibrium salt solution (HBSS) and counted. The cell count was 6 × 10⁶. 7 That was the case.

[0129] As shown in Figure 22, SBTS plasmids encoding mTCR (4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2) and transposases were electroporated into autologous PBMCs using the AMAXA NUCLEOFECTOR II instrument and kit (Lonza, Basel, Switzerland). Human T cell NUCLEOFECTOR solution (300 μL) contained SBTS plasmids encoding mTCR (45 μg) and transposases (15 μg). This human T cell NUCLEOFECTOR solution was added to cells, and then a mixture of cells and DNA was added to cuvettes (100 μL per cuvette).

[0130] As shown in Figure 22, electroporated cells were cultured overnight at 37°C in wells (CM (5 mL), IL-2 (50 IU / mL), and IL-21 (30 ng / mL) per well). After >18 hours, 50 U / mL benzonase was incubated at 37°C for 1 hour. Cells were harvested, stained, and mTCR expression was measured by FACS. A mixed population of electroporated cells was obtained, containing cells expressing mTCR (mTCR+>1%) and cells not expressing mTCR.

[0131] As shown in Figure 22, after culturing at 37°C for more than 18 hours, T cells expressing the constant region of the mouse TCR (mTCR) were observed in the mixed population. + The number of mTCR cells was selectively amplified. + To selectively amplify T cells, electroporated cells (5 × 106 ) was co-cultured with irradiated PBL (1×10 8 ) in the presence of H57 Ab (Maine Biotechnology Services, Portland, ME) (250 ng / mL), IL-2 (50 IU / mL), and IL-21 (30 ng / mL).

[0132] As shown in Figure 22, cells were cultured for 13 days. During this 13-day period, 50 / 50 CM containing IL-2 (50 IU / mL) and IL-21 (30 ng / mL) was supplied to the cells every 2 - 3 days.

[0133] As shown in Figure 22, mTCR+ cells were further enriched by contacting the cells with microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) conjugated with anti-biotin antibody and biotinylated H57 antibody in an LS column (Miltenyi Biotec).

[0134] After enrichment of mTCR+ cells with H57-binding beads, the number of mTCR + T cells was further amplified using a standard REP. In the standard REP, selectively amplified mTCR + T cells (5×10 6 ) were co-cultured with irradiated PBL (5×10 8 ), OKT3 antibody (30 ng / mL), and IL-2 (3000 IU / mL).

[0135] The schedule followed was as follows: · Day 0 = Electroporation (2×10 7 total PBMC (TP53-TCR) or CD4-depleted PMBC (HUWE1-TCR)) · Day 1 = H57 REP (T175 flask; 5×10 6 cells, 1×10 8 irradiated PBMC, 250 ng / mL H57, 50 IU / mL IL-2, 30 ng / mL IL-21) Day 14 = H57 bead concentration (H57-biotin mAb (GMP), CliniMACS biotin beads, LS column) Day 15 = OKT3 REP (GREX-100 flask; 5e6 cells, 5e8 irradiated PBMCs, 30 ng / mL OKT3, 3000 IU / mL IL-2) ·Day 28 = Harvesting, phenotyping, co-culture.

[0136] Example 15 This example illustrates the total number of cells and the percentage of CD3+mTCR+ cells obtained at four time points during the method described in Example 14.

[0137] The method described in Example 14 was performed. The total number of cells in one cuvette and the percentage of CD3+ mTCR+ cells in one cuvette were measured at four time points in the method described in Example 14: after electroporation, after selective amplification with H57, after enrichment with H57-conjugated beads, and after standard REP with OKT3.

[0138] The cells were left after electroporation and H57-conjugated bead enrichment. Counting was adjusted to reflect the actual yield rather than the theoretical yield.

[0139] The results are shown in Figure 23A (total number of cells) and Figure 23B (percentage of CD3+mTCR+ cells).

[0140] Example 16 This example shows the percentage of mTCRβ+ cells measured on day 28 (after OKT3 REP) of the method described in Example 14.

[0141] The procedure described in Example 14 was performed. The percentage of mTCRβ+ cells was measured by FACS on day 28 (after OKT3 REP). The results are shown in Figures 23C-23D. Electroporation buffer alone (mock) (without TCR) and electroporated PBMCs functioned as negative controls in Figures 23C-23D.

[0142] The percentage of CD4+mTCRβ+ or CD8+mTCRβ+ cells was measured by FACS on day 28 (after OKT3 REP). The results are shown in Figure 23E.

[0143] Example 17 This example illustrates the memory cell phenotype of cells before (day 1) and after (day 28) cell number amplification as described in Example 14.

[0144] The method described in Example 14 was followed. The expression of memory phenotype markers was measured before (day 1) or after (day 28) cell number amplification. The results are shown in Figures 24A to 24D.

[0145] Example 18 This example demonstrates the specificity of T cells after the cell number amplification described in Example 14.

[0146] The procedure described in Example 14 was performed. Immature DCs were pulsed with WT HUWE1 peptide, mutant HUWE1 peptide, WT TP53 peptide, or mutant TP53 peptide at concentrations of 10, 1, or 0.1 μg / mL. Co-cultures of peptide-pulsed immature DCs and metastatic T cells were incubated overnight at 37°C. DCs pulsed with DMSO served as a control. IFN-γ secretion was measured by ELISA. The results are shown in Figures 25A-25B.

[0147] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as each reference is individually and specifically indicated as being incorporated by reference, and to the same extent as they are incorporated herein in whole.

[0148] With regard to the description of the present invention (in particular with regard to the following claims), the use of the terms “a,” “an,” “the,” and “at least one,” and similar referents, should be interpreted as covering both singular and plural forms, unless otherwise specifically stated herein or clearly inconsistent with the context. The use of the term “at least one” after the enumeration of one or more items (e.g., “at least one of A and B”) should be interpreted as meaning one item (A or B) selected from the enumerated items or any combination of two or more items (A and B), unless otherwise specifically stated herein or clearly inconsistent with the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including but not limited to”), unless otherwise specifically stated herein. The ranges of values ​​described herein are intended solely as a way of referring to each individual value within that range, unless otherwise specified herein, and each individual value is incorporated herein as if it were individually described herein. All methods described herein may be carried out in any preferred order, unless otherwise specified herein or otherwise clearly inconsistent with the context. The use of any and all examples or illustrative terms provided herein (e.g., "such as") is intended solely to better illustrate the invention and does not impose any limitation on the scope of the invention unless otherwise claimed. No terms herein should be construed as indicating any unclaimed element as essential to the practice of the invention.

[0149] Preferred embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the above description. The inventors anticipate that such variations will be used as appropriate by those skilled in the art, and they intend that the Invention may be carried out in ways different from those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is encompassed by the Invention unless otherwise specifically noted herein or is clearly inconsistent with the context.

Claims

1. A method for preparing a substantially homogeneous T cell population, Human T cells are modified to express a TCR, and the TCR contains a mouse constant region; To produce a cell population containing some human T cells that express TCR and some human T cells that do not express TCR; (i) culturing a cell population in the presence of one or more cytokines, and (ii) a first antibody or its antigen-binding moiety that specifically binds to the mouse constant region of the β chain of the TCR; and Using the first antibody or its antigen-binding portion, human T cells expressing TCR are isolated from human T cells that do not express TCR. A method comprising a method wherein the population of T cells expressing TCR produced by the method is substantially homogeneous.

2. The method according to claim 1, wherein the TCR has antigen specificity for cancer antigens.

3. The method according to claim 1, wherein the TCR has antigen specificity for the viral antigen.

4. The method according to any one of claims 1 to 3, wherein one or more cytokines include one or more of IL-2, IL-7, IL-12, IL-15, and IL-21.

5. The method according to any one of claims 1 to 4, further comprising culturing a cell population in the presence of (i) one or both irradiated allogeneic feeder cells and irradiated autologous feeder cells, (ii) one or more cytokines, and (iii) a second antibody or its antigen-binding moiety that specifically binds to the human CD3 complex.

6. The method according to any one of claims 1 to 5, wherein the first antibody specifically binds to amino acid residues D2, R4, N5, T7, E101, D103, K104, W105, P106, E107, G108, S109 and P110 of the amino acid sequence of SEQ ID NO:

1.

7. The method according to any one of claims 1 to 6, wherein the first antibody comprises the amino acid sequences of both SEQ ID NO: 3 and SEQ ID NO:

4.

8. The method according to any one of claims 1 to 7, wherein the modification of human T cells to express a TCR comprises transfection, transformation, transduction, electroporation, transposons, meganucleases, zinc finger nucleases, and transcription-activating effector nucleases (TALEN), or the modification of human T cells to express a TCR using a clustered repeat short palindromic sequence repeat (CRISPR)-Cas system.

9. The method according to any one of claims 1 to 8, comprising increasing the number of T cells expressing a mouse constant region TCR by 10 to 1,000 times.

10. The method according to any one of claims 1 to 9, wherein the TCR includes a mouse variable region.

11. The method according to any one of claims 1 to 9, wherein the TCR includes a human variable region.

12. A method for producing a pharmaceutical composition for the treatment or prevention of diseases in mammals: The method comprises preparing a substantially homogeneous population of T cells according to any one of claims 1 to 11, A method for formulating the T cell population into the pharmaceutical composition.

13. The method according to claim 12, wherein the disease is a viral disease.

14. The method according to claim 12, wherein the disease is cancer.

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