Method for selectively expanding cells expressing TCRs with mouse constant regions

Abstract

To provide a method for preparing a substantially homogeneous T cell population.SOLUTION: Provided is a method comprising: modifying human T cells to express a TCR, the TCR comprising a murine constant region; producing a population of cells comprising a number of human T cells expressing the TCR and a number of human T cells not expressing the TCR; culturing the population of cells in the presence of (i) one or more cytokines and (ii) a first antibody or an antigen-binding portion thereof that specifically binds to the human CD3 complex; and separating the human T cells which express the TCR from the human T cells which do not express the TCR using the first antibody or an antigen-binding portion thereof, the population of T cells expressing TCR which is produced by the method being substantially homogeneous.SELECTED DRAWING: 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 October 5, 2017, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with federal support from the National Cancer Institute, National Institutes of Health under Project No. Z1A BC 010985. The federal government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Filed Materials The computer readable nucleotide / amino acid sequence listing, filed concurrently herewith and identified as follows, is hereby incorporated by reference in its entirety: One 8,876 byte ASCII (text) file entitled "740358_ST25.txt", dated September 24, 2018. [Background technology]

[0004] The treatment of cancer by administering cells that have been modified to express exogenous T cell receptors (TCRs) has produced positive clinical results in some patients. Nevertheless, obstacles remain to the wider success of such treatments. For example, low delivery efficiency of genes encoding exogenous TCRs may result in a low number of cells expressing exogenous TCRs. Therefore, there is an unmet need for improved methods for producing cells expressing exogenous TCRs. Summary of the Invention

[0005] An embodiment of the present invention provides a method for selectively expanding the number of T cells, the method comprising: modifying human T cells to express a TCR, wherein 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, wherein 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.

[0006] Further embodiments of the present invention provide relevant cell populations comprising selectively expanded numbers of T cells prepared according to the methods of the present invention, and pharmaceutical compositions comprising the cell populations.

[0007] Yet another embodiment of the present invention provides methods of treating or preventing cancer in a mammal, the methods comprising selectively expanding the number of T cells according to the methods of the present invention and administering to the mammal the selectively expanded number of T cells in an amount effective to treat or prevent cancer in the mammal. [Brief explanation of the drawings]

[0008] [Figure 1A] Figure 1A is a schematic diagram illustrating the nucleotide constructs encoding the human TCR α chain variable region (hVα), mouse TCR α chain constant region (mCα), synthetic linker sequence (L), human TCR β chain variable region (hVβ), and mouse TCR β chain constant region (mCβ). [Figure 1B] FIG. 1B is a schematic diagram illustrating a method for selectively expanding the number of T cells expressing mTCR, according to an embodiment of the present invention. [Figure 2]Figure 2 shows experimental data (dot plots) illustrating (i) CD3, CD4, or CD8 expression and (ii) mTCRβ expression detected by FACS in cells electroporated with anti-mutated ERBB2 mTCR or anti-mutated ERBB2IP mTCR (ERBB2mutTCR or ERBB2IPmutTCR) after standard Rapid Expansion Protocol (REP) using OKT3 Ab or selective amplification using H57 Ab. T cells electroporated with electroporation buffer alone (mock; no DNA / TCR) served as negative controls. Numbers in the dot plots represent the percentage of detected mTCR+ cells. [Figure 3A] Figures 3A and 3B are graphs showing the percentage (%) of anti-mutated ERBB2IP mTCRβ (ERBB2IPmutTCR) (A) or anti-mutated ERBB2 (ERBB2mutTCR) mTCRβ (B) cells that also express CD3, CD4, or CD8, detected after selective expansion with various concentrations (ng / mL) of H57 Ab. T cells electroporated with electroporation buffer alone (mock; no DNA / TCR) served as a negative control. Electroporated cells that underwent standard REP with OKT3 Ab instead of selective expansion 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-mutated ERBB2IP mTCRβ (ERBB2IPmutTCR) (A) or anti-mutated ERBB2 (ERBB2mutTCR) mTCRβ (B) cells that also express CD3, CD4, or CD8, detected after selective expansion with various concentrations (ng / mL) of H57 Ab. T cells electroporated with electroporation buffer alone (mock; no DNA / TCR) served as a negative control. Electroporated cells that underwent standard REP with OKT3 Ab instead of selective expansion with H57 Ab served as a positive control for nonspecific T cell proliferation. [Figure 4A] FIG. 4A is a schematic diagram illustrating a method for selectively expanding the number of T cells expressing a TCR comprising a mouse constant region (mTCR), according to an embodiment of the present invention. [Figure 4B] Figure 4B is a schematic diagram illustrating the binding of H57 Ab to the murine TCR β chain constant region (mCβ) of the TCR. Other components of the TCR include the human TCR α chain variable region (hVα), the murine TCR α chain constant region (mCα), a synthetic linker sequence (L), and the human TCR β chain variable region (hVβ). [Figure 5] Figures 5A-5D show experimental data (dot plots) illustrating CD3 expression and mouse TCR β chain (mTCRβ) expression detected by FACS in untransfected, unstained cells (A), untransfected, stained cells (B), cells electroporated with electroporation buffer only (mock) (no TCR / transposon (Tn)) (C), and cells electroporated with an SBTS plasmid encoding mTCR (4149-TCRa2b2 / pSBSO) and an SBTS plasmid (pKan-CMV-SB11) encoding the transposase described in Example 3 (D). Numbers in the dot plots represent the percentage of cells that are 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 plots) illustrating CD3 and mTCRβ expression detected by FACS in mTCR-electroporated cells that underwent selective amplification with the indicated initial number of electroporated cells (mTCR+ cells in REP with H57 Ab at the indicated concentrations (ng / mL)). Cells electroporated with electroporation buffer only (mock) (no TCR) served as a negative control. Numbers in the dot plots are the percentages 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 stained or unstained mTCR-electroporated cells that underwent a second round of amplification with H57 Ab or OKT3 Ab. Also shown are experimental data (dot plots) illustrating CD4 and CD8 expression detected by FACS in mTCR-electroporated cells that underwent a second round of amplification with OKT3 Ab. [Figure 8] Figures 8A and 8B show experimental data (dot plots) illustrating CD8 expression and anti-MART-1 TCR expression detected by FACS for untransduced (UT) cells (Figure 8B) and transduced cells (Figure 8A) before expansion (pre-rapid expansion protocol (REP)). Figure 8C shows experimental data (dot plots) illustrating CD8 expression and anti-MART-1 TCR expression detected by FACS for transduced cells of Figure 8A before expansion (pre-REP) and after dilution to approximately 5% mTCRb+ cells. [Figure 9] Figure 9 shows experimental data (dot plots) illustrating CD8 and anti-MART-1 TCR expression detected by FACS on UT cells or diluted transduced cells from Figure 8C after expansion 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-mutated ERBB2IP mTCRβ (ERBB2IPmutTCR) (A) or anti-mutated ERBB2 (ERBB2mutTCR) mTCRβ (B) cells that also express CD3, CD4, or CD8, detected after selective expansion with various concentrations (ng / mL) of H57 Ab. T cells electroporated with electroporation buffer alone (mock; no DNA / TCR) served as negative controls. 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 for UT cells or diluted transduced cells of Figure 8C after expansion with (i) 50 CU or 500 CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"), and Figure 10B is a graph showing the fold amplification achieved for UT cells or diluted transduced cells of Figure 8C after expansion 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 expansion of UT cells (Figure 11A) or cells transduced with anti-MART-1 TCR before (Figure 11B) or after (Figure 11C) four-fold dilution. [Figure 12] Figures 12A-12E show experimental data (dot plots) illustrating CD8 and mTCRb expression detected by FACS after expansion of UT cells (Figure 12E) or diluted TCR-transduced cells of Figure 11C with OKT3 and 500 CU IL-2 (Figure 12A), H57(mTCRb) and 500 CU IL-2 (Figure 12B), H57(mTCRb) and 50 CU IL-2 (Figure 12C), or H57(mTCRb) and no IL-2 (Figure 12D). [Figure 13] Figure 13A is a graph showing the percentage of transduced cells detected for UT cells or diluted transduced cells of Figure 11C after amplification with (i) 0 CU, 50 CU, or 500 CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"), and Figure 13B is a graph showing the fold amplification achieved for UT cells or diluted transduced cells of Figure 11C after amplification with (i) 0 CU, 50 CU, or 500 CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"). [Figure 14]Figures 14A-14F show experimental data (dot plots) illustrating CD8 and mTCRb expression detected by FACS before expansion (Figure 14A) or after expansion of transduced cells with OKT3 (30 ng / ml) (Figure 14B) or 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] FIG. 15 is a graph showing the fold amplification 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 and mTCRb expression, as detected by FACS, of mTCR-transduced cells that underwent a first round of amplification with H57 after a second round of amplification. The second round of amplification was performed with 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 fold amplification achieved for mTCR-transduced cells after a second round of expansion of mTCR-transduced cells that had undergone a first round of expansion with H57. The second round of expansion 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 plots) illustrating CD3 and mTCRb expression detected by FACS the day after electroporation of PBMCs from donors 1 and 2 with 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2. Unstained PBMCs, untransfected PBMCs, and PBMCs electroporated 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 by PBMCs from donors 1 and 2 transposed with 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2 and subjected to a first round of amplification with H57 (Figure 19B). Unstained PBMCs (Figure 19A) and PBMCs electroporated with electroporation buffer only (mock) (no TCR) (Figure 19B) served as negative controls. [Figure 20] Figures 20A-20B show experimental data (dot plots) illustrating CD3 and mTCRb expression by PBMCs from donors 1 and 2 transfected with 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2 and subjected to a first round of amplification with H57 followed by amplification with standard REP (Figure 20B). Unstained PBMCs (Figure 20A) and PBMCs electroporated with electroporation buffer only (mock) (no TCR) (Figure 20B) served as negative controls. [Figure 21]Figure 21A is a graph showing the concentration of IFN-γ secreted by (i) DCs pulsed with DMSO, WT HUWE1 peptide, or mutant (mutated) HUWE1 peptide after co-culture with (ii) cells from donor 1 transferred with 4149HUWE1-TCR1. DCs cultured alone served as controls. Mean ± SEM; n = 3 technical replicates. Figure 21B is a graph showing the concentration of IFN-γ secreted by (i) DCs pulsed with DMSO, WT TP53 peptide, or mutated TP53 peptide after co-culture with (ii) cells from donor 1 transferred with 4149-TP53-TCRa2b2. DCs cultured alone served as controls. Mean ± SEM; n = 3 technical replicates. Figure 21C is a graph showing the concentration of IFN-γ secreted by (i) DCs pulsed with DMSO, WT HUWE1 peptide, or mut HUWE1 peptide after coculture with (ii) cells from donor 2 transferred with 4149-HUWE1-TCR1. DCs cultured alone served as controls. Mean ± SEM; n = 3 technical replicates. Figure 21D is a graph showing the concentration of IFN-γ secreted by (i) DCs pulsed with DMSO, WT TP53 peptide, or mut TP53 peptide after coculture with (ii) cells from donor 2 transferred with 4149-TP53-TCRa2b2. DCs cultured alone served as controls. Mean ± SEM; n = 3 technical replicates. [Figure 22] FIG. 22 is a schematic diagram illustrating a method for selectively expanding the number of T cells expressing a TCR comprising a mouse constant region (mTCR), 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 (open bars), after selective amplification with H57 (striped bars), after enrichment with H57-coupled 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 run in parallel so that data shown are mean values ​​+ / - 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 (open bars), after selective amplification with H57 (striped bars), after enrichment with H57-coupled 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 run in parallel so that data shown are mean values ​​+ / - SEM (n=3). Figures 23C-23D are graphs showing the percentage of 4149-HUWE1-TCR1 translocated (mTCR+) (Figure 23C) or 4149-TP53-TCRa2b2 translocated (mTCR+) (Figure 23D) cells measured at day 28 (after OKT3 REP) of the method described in Example 14. PBMCs electroporated with electroporation buffer only (mock) (no TCR) served as negative controls. Mock is labeled (1), and TCR translocated cells are labeled (2). Triplicate cuvettes were run in parallel so that data shown are mean values ​​+ / - SEM (n=3). Figure 23E is a graph showing the percentage of CD4+mTCRβ+ cells (open bars) or CD8+mTCRβ+ cells (closed bars) measured on day 28 (after OKT3 REP) of the method described in Example 14. TCRs were 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2. Triplicate cuvettes were run in parallel so that data shown are mean + / - SEM (n=3). [Figure 24]Figure 24A is a graph showing the percentage of cells positive for the indicated markers measured before (day 1) (open bars) or after (day 28) (black bars) cell expansion. 4149-HUWE1-TCR1 cells were transferred. Figure 24B is a graph showing the percentage of mTCRβ cells with the indicated phenotypes measured before (day 1) (open bars) or after (day 28) (black bars) cell expansion. 4149-HUWE1-TCR1 cells were transferred. The phenotypes are central memory T (TCM) cells, memory stem T cells (TSCM cells), naive T cells (TN), effector memory T cells (TEM), and effector memory RA T cells (TEMRA). Figure 24C is a graph showing the percentage of cells positive for the indicated markers measured before (day 1) (open bars) or after (day 28) (closed bars) cell expansion. 4149-TP53-TCRa2b2 cells were transferred. Figure 24D is a graph showing the percentage of mTCRβ cells with the indicated phenotypes measured before (day 1) (open bars) or after (day 28) (closed bars) cell expansion. 4149-TP53-TCRa2b2 cells were transferred. [Figure 25] Figure 25A is a graph showing the concentration of IFN-γ secreted by DCs pulsed with DMSO or 10, 1, or 0.1 μg / mL of WT HUWE1 peptide (open bars) or mutated HUWE1 peptide (closed bars) after coculture with cells transfected with 4149-HUWE1-TCR1. Figure 25B is a graph showing the concentration of IFN-γ secreted by DCs pulsed with DMSO or 10, 1, or 0.1 μg / mL of WT TP53 peptide (open bars) or mutated TP53 peptide (closed bars) after coculture with cells transfected with 4149-TP53-TCRa2b2. DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description of the Invention Embodiments of the present invention provide methods for selectively expanding the number of T cells. The methods may include modifying human T cells to express a TCR, where the TCR includes a mouse constant region (hereinafter, "mTCR"). The methods of the present invention may provide any one or more of a variety of advantages. For example, the methods of the present invention may provide selective expansion of the number of T cells expressing mTCR relative to the number of cells that do not express mTCR. The methods of the present invention may provide a cell population comprising a higher proportion of cells expressing mTCR compared to a cell population prepared by a method that does not selectively expand the number of T cells, as described herein. Without being bound by a particular theory or mechanism, it is believed that a cell population comprising a higher proportion of cells expressing mTCR may provide improved destruction of targeted cancer cells and / or improved cancer treatment compared to a cell population comprising a lower proportion of cells expressing mTCR.

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

[0011] The mTCR may be an exogenous TCR, i.e., a TCR that is not native (naturally occurring) to the T cell. The exogenous TCR may be a recombinant TCR. A recombinant TCR is a TCR that has been produced by recombinant expression of genes encoding one or more exogenous TCR α-, β-, γ-, and / or δ-chains. Methods for producing recombinant TCRs are known in the art.

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

[0013] In one embodiment, the mTCR may comprise the amino acid sequence of the variable region of a TCR comprising the CDRs described above. In this regard, the TCR may comprise an α chain variable region and a β chain variable region.

[0014] In embodiments of the invention, the mTCR further comprises a mouse constant region in addition to the variable regions or CDRs 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," when referring to a TCR or any component of a TCR described herein (e.g., complementarity determining regions (CDRs), variable region, constant region, alpha chain, and / or beta chain), refers to a TCR (or component thereof) derived from a mouse, i.e., a TCR (or component thereof) that is of mouse T cell origin or was once expressed by a mouse T cell.

[0015] An mTCR may comprise a TCR α chain comprising a variable region and a constant region, and a TCR β chain comprising a variable region and a constant region. Hereinafter, the α chain variable region and the β chain variable region are collectively referred to as the "variable region" of the TCR. Hereinafter, the α chain constant region and the β chain constant region are collectively referred to as the "constant region" of the TCR.

[0016] In an embodiment of the invention, the mTCR is a murine TCR. A murine TCR may comprise polypeptide chains that are entirely derived from a mouse. In this regard, a murine TCR may comprise a murine variable region and a murine constant region. Examples of murine 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 invention, the mTCR is a chimeric or hybrid TCR composed of amino acid sequences derived from TCRs from two different mammalian species, i.e., a mouse and a non-mouse species. For example, the mTCR may comprise a human variable region and a mouse constant region. Examples of chimeric TCRs comprising a human variable region and a mouse constant region are disclosed in Patent Application Nos. PCT / US2016 / 050875, PCT / US2017 / 044615, PCT / US2017 / 027865, U.S. Patent Application Publication Nos. 2013 / 0274203, 2017 / 0145070, and 2016 / 0152681; and U.S. Patent No. 8,785,601.

[0018] In an embodiment of the invention, the mTCR is an antigen-specific TCR. As used herein, the phrases "antigen-specific" and "antigen specificity" mean that the mTCR is capable of specifically binding to and immunologically recognizing an antigen or an epitope thereof, such that binding of the mTCR to the antigen or an epitope thereof elicits an immune response.

[0019] The antigen recognized by the antigen-specific mTCR can be any antigen characteristic of a disease. For example, the antigen can 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, such as 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 or overexpressed solely or primarily by tumor or cancer cells, such that the antigen is associated with a tumor or cancer. A cancer antigen may also be expressed by normal, non-tumor, or non-cancerous cells. However, in such cases, expression of the cancer antigen by normal, non-tumor, or non-cancerous cells is not as robust as expression by tumor or cancer cells. In this regard, tumor or cancer cells may overexpress the antigen or express the antigen at a significantly higher level than expression by normal, non-tumor, or non-cancerous cells. A cancer antigen may also be expressed by cells in different states of development or maturation. For example, a cancer antigen may also be expressed by cells in embryonic or fetal stages, which are not normally found in adult hosts. Alternatively, a cancer antigen may also be expressed by stem or progenitor cells, which are not normally found in adult hosts. Cancer antigens are known in the art and include, for example, mesothelin, 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, MAGE-3, and the like.

[0021] In one embodiment, the cancer antigen is a neoantigen. Neoantigens are tumor- or cancer-specific antigens generated from genetic mutations that occur in tumor or cancer cells during neoplastic transformation. Neoantigens can be patient-specific. In a preferred embodiment, the neoantigen is an immunogenic neoantigen.

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

[0023] The disease associated with or characteristic of the antigen recognized by the antigen-specific mTCR can be any disease, for example, the condition can be a cancer or a viral disease, as discussed herein.

[0024] The cancer can be any cancer, including acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic bone marrow cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin's lymphoma, hypopharyngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's 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, ureter cancer, and bladder cancer.

[0025] For purposes of this specification, "viral disease" refers to a disease that can be transmitted from person to person or organism to organism and is caused by a virus. In embodiments of the present invention, the 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, the 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, or arenavirus.

[0026] The viral disease can be, for example, influenza, pneumonia, herpes, hepatitis, hepatitis A, hepatitis B, hepatitis C, chronic fatigue syndrome, sudden acute respiratory syndrome (SARS), gastroenteritis, enteritis, carditis, encephalitis, bronchiolitis, respiratory papillomatosis, meningitis, HIV / AIDS, and mononucleosis.

[0027] In an embodiment of the invention, the method includes modifying human T cells to express an mTCR. T cells can be isolated or purified. As used herein, the term "isolated" means removed from its natural environment. As used herein, the term "purified" means increased purity, and "purity" is a relative term and should not necessarily be construed as absolute purity. "Purified" T cells refer to T cells that have been separated from other natural components such as tissues, cells, proteins, nucleic acids, etc.

[0028] The T cells can be any T cells, such as cultured T cells, e.g., primary T cells, or T cells from a cultured T cell line, e.g., Jurkat, SupT1, etc., or T cells obtained from a non-mouse mammal. If obtained from a non-mouse mammal, the T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, spleen, or other tissues or fluids. The cells can also be enriched or purified. Preferably, the T cells are human T cells. The T cells are CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + The T cells can be of any type and at any stage of development, including, but 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. T cells can be CD8 + It may be a T cell or a CD4+ T cell.

[0029] The method may include modifying human T cells to express the mTCR, and obtaining expression of the mTCR by the human T cells, using any technique suitable for introducing the mTCR or a nucleic acid encoding the mTCR into the human T cells. Such techniques are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual, 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 a transposon, a lentiviral vector, or a retroviral vector. 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 can 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 transcription activator-like effector nucleases (TALENs).

[0030] The method can further include producing a cell population comprising some human T cells that express the mTCR and some human T cells that do not express the mTCR. Modifying human T cells to express the mTCR can be done with varying efficiency. Thus, modifying human T cells to express the mTCR can result in a mixed population of cells that includes cells that express the mTCR and cells that do not express the mTCR.

[0031] The method may further comprise 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, wherein the antibody specifically binds to the murine constant region of the mTCR so as to selectively expand the number of T cells that express the mTCR relative to the number of T cells that do not express the mTCR (also referred to herein as "selective amplification").

[0032] The irradiated feeder cells may include any irradiated feeder cells suitable for expanding the number of T cells. In an embodiment of the present 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 to be used is not limited and can be selected by those skilled in the art depending on various factors such as the intended use and the desired number of cells to be obtained. For example, 2 × 10 7 Irradiated feeder cells in multiples of 1 x 10 may be useful for expansion in small-scale studies. 8 Multiples of 5 x 10 irradiated feeder cells may be useful for medium-scale amplification. 8 Multiples of irradiated feeder cells are used for large-scale amplification (e.g., clinical production) (typically up to approximately 1.5 x 10 10 For example, the method may be useful for detecting approximately 1×10 9 ~Approx. 4×10 9 Allogeneic and / or autologous feeder cells, preferably about 2×10 9 ~Approx. 3×10 9 Allogeneic and / or autologous feeder cells may be used.

[0033] The one or more cytokines may include any one or more cytokines suitable for expanding the number of T cells. In an embodiment of the present invention, the one or more cytokines include any one or more of interleukin (IL)-2, IL-7, IL-12, IL-15, and IL-21. Includes.

[0034] The method may further include culturing the cell population in the presence of an antibody or antigen-binding portion thereof, 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 of 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., rabbit, goat, horse, chicken, hamster, human, etc. Alternatively, the antibody may be a genetically engineered antibody, e.g., a humanized antibody or chimeric antibody. The antibody may be in monomeric or multimeric form. The antibody or antigen-binding fragment thereof may have any level of affinity or avidity for the mouse constant region of the mTCR.

[0035] In embodiments of the invention, an antibody comprises two polypeptide chains (heavy and light), each of which comprises a variable region comprising complementarity determining regions (CDRs) 1, 2, and 3 of the antibody. An 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 invention, an antibody comprises a heavy chain variable region and a light chain variable region.

[0036] An antigen-binding portion of an antibody can be any portion that has at least one antigen-binding site. In an embodiment of the present invention, the antigen-binding portion can comprise the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of an antibody. In another embodiment of the present invention, the antigen-binding portion can comprise the heavy chain variable region and light chain variable region of an antibody. The antigen-binding portion of an antibody can be a Fab fragment (Fab), a F(ab')2 fragment, a Fab' fragment, an Fv fragment, a single-chain variable region fragment (scFv), a disulfide-stabilized variable region fragment (dsFv), scFv2CH3, scFv4, scFv3, scFv2, scFv-Fc, or (scFv)2. Single-chain variable region fragments (scFvs), which are fusion proteins comprising the variable (V) domain of an antibody heavy chain linked to the variable (V) domain of an antibody light chain via a synthetic peptide, can be produced using conventional recombinant DNA technology techniques. Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology. However, the antigen-binding portion of an antibody is not limited to these exemplary types of antigen-binding portion. Unless otherwise specified, antibodies and antigen-binding portions thereof will hereinafter be collectively referred to as "antibodies."

[0037] The antibody can be any antibody that specifically binds to the murine constant region of the mTCR. In embodiments of the invention, the antibody specifically binds to the murine 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 referred to as the CD3 complex.

[0038] The antibody may specifically bind to the murine constant region of the mTCR α chain or the murine constant region of the mTCR β chain. In a preferred embodiment, the antibody specifically binds to the murine constant region of the mTCR β chain. The murine constant region of the mTCR β chain to which the antibody specifically binds may comprise or consist of the amino acid sequence of SEQ ID NO: 1 (the amino acid sequence of the full-length murine constant region of the mTCR β chain). The antibody may specifically bind to any part of the murine constant region of the mTCR β 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 of the full-length murine constant region of the mTCR β chain). In an embodiment of the 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 mTCR are commercially available. For example, an antibody that specifically binds to the mouse constant region of the β chain of mTCR is the H57 antibody (also called H57-597) (available from Biolegend, San Diego, CA). H57 is an Armenian hamster IgG antibody, 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 invention, an antibody comprises a heavy chain variable region and a light chain variable region. For example, an antibody (Ab) can comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 3 (the variable region of the H57 Ab heavy chain) or SEQ ID NO: 4 (the variable region of the H57 Ab light chain), or both SEQ ID NOs: 3 and 4. Preferably, an antibody comprises the amino acid sequences of both SEQ ID NOs: 3 and 4. In embodiments of the invention, an antibody comprises complementarity determining regions (CDRs) 1, 2, and 3 of the H57 Ab heavy chain of SEQ ID NO: 3, and CDR1, 2, and 3 of the H57 Ab light chain of SEQ ID NO: 4.

[0041] In embodiments of the invention, the antibody comprises a heavy chain and a light chain comprising the variable regions described above. For example, the antibody can comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 5 (H57 Ab heavy chain) or SEQ ID NO: 6 (H57 Ab light chain), or both SEQ ID NOs: 5 and 6. Preferably, the antibody comprises the amino acid sequence of both SEQ ID NOs: 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 advantageously expands the number of T cells expressing mTCR selectively relative to the number of T cells not expressing mTCR. In this regard, the methods of the invention can advantageously provide a cell population containing a higher proportion of cells expressing mTCR compared to methods of expanding cell number that do not use an antibody that specifically binds to the mouse constant region of the TCR. In embodiments of the invention, the methods increase the number of T cells expressing mTCR by about 5-fold or less to about 4,000-fold or more. For example, the methods of the present invention can increase the number of mTCR-expressing cells by about 5-fold to about 4,000-fold, about 100-fold to about 3,500-fold, about 1,000-fold to about 3,000-fold, about 1,500-fold to about 2,500-fold, about 10-fold to about 1,000-fold, about 50-fold to about 850-fold, about 100-fold to about 900-fold, about 150-fold to about 850-fold, about 200-fold to about 800-fold, about 250-fold to about 750-fold, about 300-fold to about 700-fold, about 350-fold to about 650-fold, or about 400-fold to about 600-fold. For example, the methods of the present invention can increase the number of mTCR-expressing cells by about 10-fold, about 50-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1,000-fold, or a range of 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 methods of the present invention can be highly variable and donor-dependent.

[0043] In embodiments of the present invention, the method produces a selectively expanded cell population, wherein about 10% to about 95% of the cells in the selectively expanded population express a TCR comprising a mouse constant region. In this regard, the method may be used to produce a selectively expanded cell population in which about 10% to about 95%, about 15% to about 90%, about 20% to about 85%, about 25% to about 80%, or about 30% to about 75% of the cells in the selectively expanded population express a TCR comprising a mouse constant region. The method may produce a selectively expanded cell population in which about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or a range between any two of the foregoing values ​​of the cells in the selectively expanded population express mTCR.

[0044] The methods of the present invention can advantageously produce any number of mTCR-expressing T cells that may be suitable for any of a variety of uses. In embodiments of the present invention, the methods produce approximately 1 x 10 mTCR-expressing T cells. 6 ~Approx. 1×10 11 For example, the method of the present invention can produce approximately 1 x 10 T cells expressing mTCR in a small vessel (e.g., a T25 flask). 6 ~Approx. 1×10 7 The method of the present invention can produce approximately 5 x 10 T cells expressing mTCR in a larger vessel (e.g., a T175 flask). 6 ~Approx. 3×10 7 The methods of the present invention can produce approximately 1 x 10 T cells expressing mTCR in other containers (e.g., GREX flasks available from Wilson Wolf Manufacturing, New Brighton, MN). 9 ~Approx. 1×10 10 Approximately 1 x 10 T cells expressing mTCR can be produced. 6 ~Approx. 1×10 10 A population of T cells expressing mTCR may be useful for small-scale screening experiments. A larger number, e.g., about 1.5 x 10, may be useful for small-scale screening experiments. 10 These cells may also be obtained using the methods of the invention, for example, for clinical applications. The number of mTCR-expressing T cells produced by the methods of the invention can be highly variable and donor-dependent.

[0045] The method may comprise performing one or more selective amplifications of the number of mTCR-expressing cells, or multiple selective amplifications of the number of mTCR-expressing cells. In embodiments of the invention, the method comprises performing one or more selective amplifications, as described herein with respect to other aspects of the invention, followed by one or more non-selective amplifications of the number of cells. In this regard, the method may further comprise culturing human T cells in the presence of (i) one or both of irradiated allogeneic feeder cells and irradiated autologous feeder cells, (ii) one or more cytokines, and (iii) an antibody that specifically binds to the human CD3 complex, or an antigen-binding portion thereof (also referred to herein as "non-selective amplification"). Expanding the number of T cells can be achieved by any of a number of methods, as described, for example, in U.S. Pat. No. 8,034,334; U.S. Pat. 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, the number of T cells can be non-selectively expanded using an antibody or antigen-binding portion thereof 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), with IL-2 being preferred. An example of an antibody that specifically binds to the human CD3 complex is OKT3 (available from Ortho-McNeil, Raritan, NJ).

[0046] Multiple rounds of selective amplification, or one or more rounds of selective amplification followed by one or more rounds of non-selective amplification, can increase the number of mTCRs by about 100,000-fold or more. In embodiments of the invention, the methods produce cell populations in which about 20% to about 99% of the cells in the population express TCRs comprising a mouse constant region. Multiple rounds of selective expansion, or one or more rounds of selective expansion followed by one or more rounds of non-selective expansion, can produce cell populations in which about 25% to about 95%, about 30% to about 90%, about 35% to about 85%, about 40% to about 80%, about 45% to about 75%, or about 50% to about 70% of the cells in the population express mTCRs. The method may include detecting a concentration of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or any two of the foregoing values ​​in the population. A cell population can be produced in which a range of cells express the mTCR.

[0047] In embodiments of the invention, the method further comprises separating TCR-expressing T cells from T cells that do not express the TCR using an antibody or antigen-binding portion thereof that specifically binds to the murine constant region of the TCR. In this regard, the method may comprise physically contacting a mixed population of cells, including cells that express mTCR and cells that do not express mTCR, with the antibody, such that the antibody specifically binds to the murine constant region of the TCR. The antibody may be attached to a support, e.g., beads. The method may further comprise washing the antibody and cells such that all or a portion of the cells that do not express mTCR are removed from the cells that express mTCR. The method may further comprise eluting the mTCR-expressing cells from the antibody. Exemplary 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 advantageously provide cell populations enriched for cells expressing mTCR. Accordingly, embodiments of the present invention provide cell populations comprising a number of selectively expanded T cells prepared according to any of the methods described herein. The cell population can be a heterogeneous population comprising T cells expressing mTCR along with at least one other cell (e.g., a T cell that does not express mTCR) or a cell other than a T cell (e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc.). Alternatively, the cell population can be a substantially homogeneous population, in which the population primarily comprises (e.g., consists essentially of) T cells expressing mTCR. The population can also be a clonal population of cells, in which all cells of the population are clones of a single T cell expressing mTCR, such that all cells of the population express mTCR. In one embodiment of the present invention, the cell population is a clonal population comprising T cells expressing mTCR.

[0049] The cell populations of the present invention may be isolated and / or purified. As used herein, the term "isolated" means removed from its natural environment. As used herein, the term "purified" means increased purity, and "purity" is a relative term and should not necessarily be construed as absolute purity. Purity may be, for example, at least about 50%, about 60%, about 70%, about 80%, about 90%, greater than about 95%, or about 100%.

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

[0051] Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier can be any of those conventionally used for T cells. Methods for preparing administrable compositions are known or obvious to those skilled in the art, and are described, for example, in Remington: The Science and Practice of Pharmacy, 2000; 2nd Edition, Pharmaceutical Press (2012). Preferably, a pharmaceutically acceptable carrier is one that has no detrimental side effects or toxicity under the conditions of use.

[0052] The choice of carrier can be determined by the particular 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 can include those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or intraperitoneal administration. More than one route can be used to administer the cell population of the present invention, and in certain instances, one route can provide a more immediate and effective response than another route.

[0053] Preferably, the cell population of the present invention is administered by injection, e.g., intravenously. Pharmaceutically acceptable carriers for cells for injection include, for example, normal saline (about 0.90% in water). The infusion solution may include any isotonic carrier such as about 1000 mg / L w / v NaCl, 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 lactated Ringer's 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 purposes of the present invention, the amount or dose (e.g., number of cells) administered should be sufficient to effect, e.g., a therapeutic or prophylactic response in a mammal over a reasonable time frame. For example, the dose (e.g., number of cells) should be sufficient to bind to a disease-associated antigen or to detect, treat, or prevent 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 potency of the cell population and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated.

[0055] Many assays for determining dosages are known in the art. For purposes of the present invention, an assay can be used to determine a starting dose to administer to a mammal, which assay involves comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing mTCR when a particular dose of T cells is administered to the mammal, between a set of mammals each receiving various 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 dose of the cell population of the present invention will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular cell population. Typically, the attending physician will determine the dose of cell population to treat each individual patient, taking into account 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 x 10 6 Approximately 1 x 10 cells 12 In certain embodiments, the number of cells can vary from about 1.5 x 10 to 1.5 x 10 cells or more. 10 Cells may be administered.

[0057] The cell populations produced by the methods of the invention may be useful for treating or preventing disease, e.g., cancer. Accordingly, another embodiment of the invention provides a method of treating or preventing disease in a mammal, the method comprising selectively expanding the number of T cells according to any of the methods described herein with respect to other aspects of the invention, and administering to the mammal the selectively expanded number of T cells in an amount effective to treat or prevent the disease in the mammal.

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

[0059] In embodiments of the invention, the disease is cancer. The cancer may be any of the cancers described herein with respect to other aspects of the invention.

[0060] In an embodiment of the invention, the disease is a viral disease. The viral disease may be any of the viral diseases described herein with respect to other aspects of the invention.

[0061] As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits. Preferably, the mammal is from the order Carnivora, including felines (cats) and canines (dogs). More preferably, the mammal is from the order Bovidae, including bovines (cattle) and swine (pigs), or from the order Equidae, including equidae (horses). Most preferably, the mammal is from the order Primates, Ceboids, or Simoides (monkeys), or from the order Anthropoidea (humans and apes). A particularly preferred mammal is a human.

[0062] As used herein, the terms "treatment" and "prevention," and words derived therefrom, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art will recognize as having potential benefit or therapeutic effect. In this regard, the methods of the present invention may provide any amount or level of cancer treatment or prevention in a mammal. Moreover, the treatment or prevention provided by the methods of the present invention can include 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 can include promoting tumor regression. For purposes of this specification, "prevention" can also include delaying the onset of a disease, or its symptoms or conditions.

[0063] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0064] Example 1 This example demonstrates that selectively expanding the number of T cells expressing TCRs containing a murine constant region (mTCR) increases the number of cells expressing mTCRs.

[0065] RNA encoding two TCRs, one with antigen specificity for a cancer-specific mutant ERBB2 neoantigen and the other with antigen specificity for a cancer-specific mutant ERBB2IP neoantigen, was isolated from tumor-infiltrating lymphocytes (TILs) isolated from cancer patients. cDNA was amplified from the RNA via reverse transcription, followed by PCR amplification of the cDNA copies. mTCR constructs (one per TCR) were prepared as described by 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 a synthetic linker sequence placed 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) (Deniger et al., PLoS One, 10(6):e0128151 (2015) and Singh et al., Cancer Res., 71(10):3516-27 (2011)). description).

[0066] According to an embodiment of the present invention, peripheral blood mononuclear cells (PBMCs) are electroporated with mTCR and + A schematic diagram illustrating the method for selectively expanding the number of T cells is shown in Figure 1B. As shown in Figure 1B, using the AMAXA NUCLEOFECTOR II device and kit (Lonza, Basel, Switzerland), SBTS plasmids (15 μg) encoding one of the two mTCRs and SBTS plasmids (5 μg) encoding the transposase were transfected into autologous PBMCs (2 × 10 7 After 24 hours, a mixed population of electroporated cells was obtained, including cells that expressed mTCR and cells that did not express mTCR.

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

[0068] mTCR + After selectively expanding the number of T cells, the cells were incubated with (i) anti-CD3 Ab, anti-CD4 Ab Ab, or anti-CD8 Ab and (ii) anti-mTCRβ antibody (H57). PBMCs electroporated with electroporation buffer alone (mock) (no TCR) served as negative controls.

[0069] The stained cells were counted by fluorescence-activated cell sorting (FACS). The results are shown in Figure 2. As shown in Figure 2, selective amplification by H57 Ab resulted in a higher number of anti-mutated ERBB2 mTCRs compared to OKT3. + T cell numbers and anti-mutated ERBB2IP mTCR + The number of T cells increased.

[0070] Example 2 This example shows that higher concentrations of H57 Ab resulted in a greater number of mTCR + We demonstrate that

[0071] PBMCs were electroporated with anti-ERBB2IP mTCR or anti-ERBB2 mTCR constructs as described in Example 1. T cells expressing murine TCR constant regions (mTCR +The number of HIV-1 T cells was selectively expanded with 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 expansion of mTCR+ T cell numbers, 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) (no TCR) served as a negative control. Electroporated cells that underwent standard REP with OKT3 Ab (30 ng / mL) instead of selective expansion 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, a higher concentration of H57 Ab resulted in a greater number of anti-mutated ERBB2 mTCR + and anti-mutated ERBB2IP mTCR + cells were obtained.

[0074] Example 3 This example demonstrates a method to selectively expand 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 ovarian cancer patient 4149. cDNA was amplified from the RNA by reverse transcription, and the cDNA copy was then amplified by PCR. mTCR constructs were prepared by fusing the human TCR-α VJ region to the mouse TCR-α constant chain and the human TCR-β VDJ region to the mouse TCR-β constant chain, with a synthetic linker sequence located between the α and β chains (Figure 4B). mTCR constructs were synthesized and cloned into SBTS plasmids.

[0076] According to an embodiment of the present invention, PBMCs are electroporated with mTCR and + A schematic diagram illustrating the method for selectively expanding the number of T cells is shown in Figure 4A. As shown in Figure 4A, autologous PBMCs were obtained from patients and cryopreserved. Cryopreserved PBMCs (200 g) were thawed at 20-23°C for 10 minutes. PBMCs were then resuspended in 50 / 50 medium (3 × 10 6 The SBTS plasmid encoding mTCR and the SBTS plasmid encoding transposase were transfected with AMAXA NUCLEOFECTOR Autologous PBMCs were electroporated using a 1.2L device and kit (Lonza, Basel, Switzerland). Human T cell nucleofecator solution (300 μL) contained 45 μg of SBTS plasmid encoding mTCR and 15 μg of SBTS plasmid encoding transposase. This human T cell nucleofecator solution was added to cuvettes (100 μL / 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 added and incubated for 1 hour at 37°C. A mixed population of electroporated cells was obtained, including cells expressing mTCR and cells not expressing mTCR.

[0077] As shown in Figure 4A, after >18 hours of culture at 37°C, T cells expressing murine TCR constant regions (mTCR + The number of mTCR (mTCR T cells) was selectively amplified. + To selectively expand T cells, mTCR was expressed 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 ) irradiated peripheral blood lymphocytes (PBLs) (1 × 10 8 ) and co-cultured with

[0078] mTCR + After selective expansion of T cell numbers, mTCR+ The number of T cells was further expanded using a standard rapid expansion protocol (REP) (see Example 5). In standard REP, selectively expanded mTCR + T cells (5×10 6 ) were cultured in irradiated PBLs (5 × 10 8 The cells were co-cultured with IL-2 (3000 IU / mL), 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. Negative controls included untransfected autologous PBMCs and PBMCs electroporated with electroporation buffer only (mock). Electroporated cells were stained with anti-CD3 and anti-mTCRβ antibodies (H57). Unstained cells were stained with anti-CD3 and anti-mTCRβ antibodies (H57). Untransfected autologous PBMC served as an additional negative control.

[0081] The day after electroporation, the stained cells were counted by FACS as described in Example 4 without undergoing selective amplification. (neg) Gated on cells. PI is a stain for dead cells. (neg) The 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 the total cells) expressed mTCR after electroporation without undergoing selective expansion.

[0083] Example 4 This example demonstrates that selective expansion of electroporated T cells with the H57 antibody selectively increases the number of cells expressing TCRs containing murine constant regions in a mixed population.

[0084] Autologous PBMCs were electroporated with SBTS plasmids encoding mTCR and transposase as described in Example 3. Various starting numbers of mTCR+ T cells (1 × 10 5 , 5×10 5 , or 10 x 10 5 ) 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 5 × 10 based on the initial frequency. 5 mTCR+ T cells were aligned. For example, 5% mTCR expression corresponds to a total of 1 × 10 7 Transition 5 x 10 cells into the amplification protocol 5 The objective of this study was to achieve mTCR+ T cells. Electroporation buffer alone (mock) and electroporated PBMCs served as negative controls. Electroporated, selectively expanded cells were stained with anti-CD3 and anti-mTCRβ antibodies (H57).

[0085] The stained cells were counted by FACS. Gating was performed on lymphocytes / PI (neg) cells by FACS. The results are shown in Figure 6 and Tables 1-2. As shown in Figure 6 and Tables 1-2, selective amplification by H57 Ab resulted in the proliferation of mTCR in the population. + The number of T cells increased.

[0086] [Table 1-1]

[0087] [Table 1-2]

[0088] [Table 2]

[0089] Example 5 In this example, the first amplification with H57 Ab followed by the second amplification with OKT3 Ab resulted in mTCR + Further increases in cell numbers are demonstrated.

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

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

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

[0093] The percentage of viable cells, total number of cells, fold change in number of cells achieved by the second round of expansion, number of mTCR+ cells, and fold change in mTCR+ cells achieved by the second round of expansion were also measured and compared for cells that underwent a second round of expansion with OKT3 Ab and H57 Ab. The results are shown in Table 3. As shown in Table 3, the second round of expansion with OKT3 Ab after the first round of expansion with H57 Ab resulted in a significant increase in mTCR+ cells. + The number of mTCR cells further increased after the second round of amplification with OKT3 Ab compared to the second round of amplification with H57 Ab. + The number of cells increased significantly.

[0094] [Table 3]

[0095] Example 6 This example demonstrates that selectively expanding the number of T cells expressing mTCR increases the number of cells expressing mTCR.

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

[0097] [Table 4]

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

[0099] The cell numbers in the 5% mTCRb+ starting population in Figure 8C after expansion with (i) OKT3 (50 ng / ml) or H57 (50 ng / ml), and (ii) either 500 or 50 CU / IL-2 are shown in Figures 9 and 10A-10B. Although H57 amplified approximately 1500-2300-fold, there was no selective expansion of the number of cells in the mTCRb+ cell population. Alternatively, H57 amplified fewer cells (500-800-fold), but they did show a 4-8-fold increase in the percentage of mTCRb+ cells. Overall, the use of H57 in expansion resulted in more mTCRb+ cells.

[0100] Example 7 This example demonstrates that selectively expanding the number of T cells expressing mTCR increases the number of cells expressing mTCR.

[0101] PBLs were transduced with a retroviral vector encoding the murine F5 TCR as described in Example 6. Expression of CD8 and mTCRb by the transduced cells (starting cell population) before expansion with 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 4-fold to 14% mTCRb+ (Figure 11C).

[0102] The numbers of transduced cells and UT cells were expanded (i) with no IL-2, 50 CU IL-2, or 500 CU IL-2, and (ii) with OKT3 Ab or H57 Ab. The results are shown in Figures 12A-12E and 13A-13B.

[0103] After expansion, the OKT3 population showed no selective expansion of the overall mTCRb+ cell population, with approximately equal percentages (5%) of CD8+ and CD8-transduced cells. In comparison, both H57-amplified populations (500 or 50 CU / IL-2, respectively) showed selective mTCRb+ expansion of 31.2% and 59.1%, respectively. H57 expansion with 50 CU / IL-2 showed nearly two-fold greater expansion 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-fold in the presence of 500 CU / IL-2, but H57 amplification with 50 CU / IL-2 increased the number by approximately 3500-fold (more than double that of the other conditions tested).

[0105] Example 8 This example demonstrates that selectively expanding the number of T cells expressing mTCR increases the number of cells expressing mTCR.

[0106] PBLs were transduced with a retroviral vector encoding the murine F5 TCR as described in Example 6. Expression of CD8 and mTCRb by transduced cells (pre-REP) before amplification with H57 or OKT3 is shown in Figures 14A-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 can reduce the titer by 10-fold without any adverse effect on the selective mTCRb+ expansion. The overall fold expansion was also similar among all groups tested. Preferential expansion of CD8- cells after expansion with H57 was also observed.

[0108] Example 9 This example shows that the number of cells that underwent a first round of amplification with H57 in Example 8 was further amplified by a second round of amplification with either OKT3 or H57.

[0109] The mTCR-transduced cells that underwent amplification with H57 Ab in Example 8 were subjected to a second round of amplification with OKT3 or H57 to determine whether further enrichment of mTCRb+ cells could be achieved. The amplification conditions were identical to those described in Example 8.

[0110] The results are shown in Figures 16A-16E and 17. A second round of amplification with OKT3 did not further enrich for mTCRb+ cells, but did further expand the number of cells by 1000-fold. A second round of amplification with H57 did not add any further enrichment for mTCRb+ cells and may have resulted in a slight decrease in mTCRb+ cells at 500 and 50 ng / ml. The fold expansion of the population that underwent a second round of amplification with H57 was low, ranging from 200- to 500-fold.

[0111] Example 10 This example demonstrates that the mutation-specific TCR is expressed by the transposon 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 (200 g) for 10 min at 20-23 °C. The PBMCs were placed in 50 / 50 medium (3 × 10 6 The cells were incubated at 37°C for 2 hours. SBTS plasmids encoding one of the two mTCRs and a transposase-encoding SBTS plasmid were electroporated into autologous PBMCs using an AMAXA NUCLEOFECTOR II device and kit (Lonza, Basel, Switzerland). The two mTCRs were (1) 4149-HUWE1-TCR1 (class I) and (2) 4149-TP53-TCRa2b2 (class II). The human T cell NUCLEOFECTOR solution (300 μL) contained SBTS plasmids encoding the mTCRs (45 μg) and SBTS plasmids encoding the transposase (15 μg). After a 2-hour resting period, nonadherent PBMCs were harvested by centrifugation (200 g) for 10 minutes at 20–23°C, and the medium was removed and replaced with human T cell NUCLEOFECTOR solution containing the SBTS plasmids (100 μL 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 added and incubated for 1 hour at 37°C. A mixed population of electroporated cells was obtained, including cells that expressed mTCR and cells that did not express mTCR.

[0113] The day after electroporation, electroporated cells were incubated with (i) anti-CD3 The cells were stained with (i) anti-mTCRβ Ab and (ii) anti-mTCRβ Ab. Unstained PBMCs, non-transfected PBMCs, and PBMCs electroporated with electroporation buffer only (mock) (no TCR) served as negative controls. Expression of CD3 and mTCR by electroporated cells was measured by FACS (Gate: Lymphocytes\Live Cells (PI-)). The results are shown in Figure 18. As shown in Figure 18, the transposon expressed the mutant-specific TCR the day after electroporation.

[0114] Example 11 This example demonstrates that a single round of amplification with H57 results in a selective expansion of the number of mTCR+ T cells.

[0115] PBMCs from donors 1 and 2 were electroporated as described in Example 10. As shown in Figure 4A, after more than 18 hours of culture at 37°C, T cells expressing the murine TCR constant region (mTCR + The number of mTCR (mTCR T cells) was selectively increased. + To selectively expand the number of T cells, mTCR + T cells (5×10 5 ) to irradiated PB L(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).

[0116] Selectively expanded cells were stained and assessed by FACS as described in Example 10. Unstained PBMCs (FIG. 19A) and PBMCs electroporated with electroporation buffer only (mock) (no TCR) (FIG. 19B) served as negative controls. The results are shown in FIGS. 19A-19B. In FIGS. 19A-19B, cell numbers were determined 14 days after a single round of expansion with H57 from one T175 flask. As shown in FIGS. 19A-19B, a single round of expansion with H57 resulted in a selective expansion in the number of mTCR+ T cells.

[0117] Example 12 This example demonstrates that further expansion of the number of cells that have undergone a first round of expansion with H57, followed by a second round of expansion with OKT3 in gas-permeable flasks, results in a significant expansion of the number of TCR-transferred T cells.

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

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

[0120] [Table 5]

[0121] [Table 6]

[0122] The results are shown in Figures 20A-20B. As shown in Figures 20A-20B, further expansion of the number of cells that underwent a first round of expansion with H57 followed by a second round of expansion with OKT3 in gas-permeable flasks resulted in a significant expansion of the number of TCR-transferred T cells.

[0123] Example 13 This example demonstrates that TCR-transferred T cells selectively expanded by the H57 antibody are specific for the cognate mutant neoantigen.

[0124] PBMCs from donors 1 and 2 were electroporated as described in Example 10. The number of electroporated cells was selectively expanded with 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 10 peptide-pulsed immature DCs 5 Co-cultures of T cells were incubated overnight at 37°C. DCs were cultured alone, 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 expanded with H57 antibody recognized the cognate mutant neoantigen.

[0127] Example 14 This example demonstrates a method to selectively expand the number of T cells expressing mTCR.

[0128] PBMCs are electroporated with mTCR and transfected with mTCR according to an embodiment of the invention. + A schematic illustrating the method for selectively expanding the number of T cells is shown in Figure 22. As shown in Figure 22, autologous PBMCs were obtained from patients and cryopreserved. A portion of the PBMCs was depleted of CD4+ cells using an LD column. The cryopreserved PBMCs were thawed in complete medium (CM) and centrifuged at 175g for 10 minutes at approximately 22°C. The cells were washed in Hank's balanced salt solution (HBSS) and counted. The cell number was 6 x 10 7 It was.

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

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

[0131] As shown in Figure 22, after more than 18 hours of culture at 37°C, T cells expressing the murine TCR constant region (mTCR + The number of mTCR (mTCR T cells) was selectively increased. + To selectively expand T cells, electroporated cells (5 × 10 6 ) were cultured in irradiated PBLs (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] Cells were cultured for 13 days, as shown in Figure 22. During this 13-day period, cells were fed every 2-3 days with 50 / 50 CM containing IL-2 (50 IU / mL) and IL-21 (30 ng / mL).

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

[0134] After enrichment of mTCR+ cells with H57-conjugated beads, mTCR + The number of T cells was further expanded using standard REP, in which selectively amplified mTCR + T cells (5×10 6 ) were cultured in irradiated PBLs (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 x 10 7 of total PBMCs (TP53-TCR) or CD4-depleted PBMCs (HUWE1-TCR) Day 1 = H57 REP (T175 flask; 5 x 10 6 cells, 1 x 10 8 of irradiated PBMCs, 250ng / mL H57, 50IU / mL IL-2, 30ng / mL IL-21) Day 14 = H57 bead enrichment (H57-biotin mAb (GMP), CliniMACS biotin beads, LS column) Day 15 = OKT3 REP (GREX-100 flask; 5e6 cells, 5e8 irradiated PBMCs, 30ng / mL OKT3, 3000IU / mL IL-2) ·Day 28 = Harvesting, phenotyping, co-culture.

[0136] Example 15 This example demonstrates the total number of cells and 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 during the method described in Example 14: after electroporation, after selective amplification with H57, and after amplification with H57-bound beads. Measurements were taken after concentration and after standard REP with OKT3.

[0138] Cells were left after electroporation and H57-coupled bead enrichment. Counts were adjusted to reflect actual yields rather than theoretical yields.

[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 demonstrates the percentage of mTCRβ+ cells measured at day 28 (post OKT3 REP) of the method described in Example 14.

[0141] The method 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 only (mock) (no TCR) and electroporated PBMCs served 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), and the results are shown in Figure 23E.

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

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

[0145] Example 18 This example demonstrates the specificity of T cells after expansion of cell numbers as described in Example 14.

[0146] The method 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. Cocultures of peptide-pulsed immature DCs and transferred 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 herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and to the same extent as if each reference was set forth in its entirety herein.

[0148] With respect to describing the present invention (particularly with respect to the claims that follow), use of the terms "a," "an," "the," and "at least one" and similar referents should be construed to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. Use of the term "at least one" after a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including ( The terms "including" and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise indicated. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise stated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary phrases (e.g., "such as") provided herein is intended only to better illustrate the invention and does not impose a limitation on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0149] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors anticipate that those of ordinary skill in the art will employ such variations as appropriate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. 1. A method for preparing a substantially homogeneous population of T cells, comprising: modifying blood-derived human T cells to express a TCR, wherein the TCR comprises a murine constant region; producing a cell population comprising some human T cells that express said TCR and some human T cells that do not express said TCR; (i) culturing the cell population in the presence of one or more cytokines, and (ii) a first antibody, or antigen-binding portion thereof, that specifically binds to a mouse constant region of the β chain of the TCR; and using the first antibody, or antigen-binding portion thereof, to separate human T cells that express the TCR from human T cells that do not express the TCR. wherein the population of T cells expressing a TCR produced by said method is substantially homogeneous.

2. The method of claim 1, wherein the TCR has antigen specificity for a cancer antigen.

3. The method of claim 1 , wherein the TCR has antigen specificity for a viral antigen.

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

5. 5. The method of any one of claims 1 to 4, further comprising culturing the cell population in the presence of (i) one or both of irradiated allogeneic feeder cells and irradiated autologous feeder cells, and (ii) one or more cytokines.

6. 6. The method of any one of claims 1 to 5, further comprising culturing the cell population in the presence of a second antibody, wherein the second antibody specifically binds to the human CD3 complex.

7. The method of 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 of any one of claims 1 to 7, wherein the step of modifying a human T cell to express a TCR comprises a step of modifying a human T cell to express the TCR using transfection, transformation, transduction, electroporation, a transposon, a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), or a clustered repeated short palindromic repeat (CRISPR)-Cas system.

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

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

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

12. 1. A method for preparing a pharmaceutical composition for the treatment or prevention of a disease in a mammal, comprising: preparing a substantially homogeneous population of T cells according to the method of any one of claims 1 to 11, wherein the population of T cells is formulated into the pharmaceutical composition.

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

14. 13. The method of claim 12, wherein the disease is cancer.

Images