Method for selective expansion of cells expressing TCRs with murine constant regions

Abstract

The present application discloses a method for selectively expanding a plurality of T cells. The method may include: modifying human T cells to express a TCR, wherein the TCR includes a mouse constant region; generating a cell population comprising a plurality of human T cells expressing the TCR and a plurality of 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 an antigen-binding portion thereof, wherein the antibody has antigen specificity for the mouse constant region of the TCR, so as to selectively expand the number of T cells expressing the TCR over the number of T cells not expressing the TCR. The present application also discloses a related cell population, a pharmaceutical composition, and a method for treating or preventing cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 568,339, filed on October 5, 2017, which is incorporated herein by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support through the National Cancer Institute of the National Institutes of Health under Grant No. Z1A BC 010985. The government has certain rights in this invention.

[0005] Incorporation by Reference of Electronically Submitted Materials

[0006] Incorporated herein by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing filed concurrently and identified as follows: an 8,876-byte ASCII (text) file named "740358_ST25.txt" dated September 24, 2018. Background of the Invention

[0008] Treating cancer by administering cells modified to express exogenous T cell receptors (TCRs) has produced positive clinical results in some patients. However, there are still obstacles to the more extensive success of such therapies. For example, the low delivery efficiency of genes encoding exogenous TCRs may result in a lower number of cells expressing exogenous TCRs. Therefore, the demand for improved methods for producing cells expressing exogenous TCRs has not yet been met.

[0009] Brief description of the invention

[0010] Embodiments of the present invention provide a method for selectively expanding a plurality of T cells, the method comprising: modifying human T cells to express a TCR, wherein the TCR comprises a mouse constant region; generating a cell population comprising a plurality of human T cells expressing the TCR and a plurality of 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 an antigen-binding portion thereof, wherein the antibody has antigenic specificity for the mouse constant region of the TCR, so as to selectively expand the number of T cells expressing the TCR over the number of T cells not expressing the TCR.

[0011] Additional embodiments of the present invention provide related cell populations comprising a plurality of T cells selectively expanded according to the methods of the present invention, and pharmaceutical compositions comprising the cell populations.

[0012] Yet another embodiment of the present invention provides a method for treating or preventing cancer in a mammal, the method comprising selectively expanding a plurality of T cells according to the method of the present invention, and administering the selectively expanded plurality of T cells to the mammal in an amount effective for treating or preventing cancer in the mammal.

[0013] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0014] Figure 1A Schematic diagram illustrating the nucleotide construct encoding the human TCR α chain variable region (hVα), the mouse TCR α chain constant region (mCα), the synthetic linker sequence (L), the human TCR β chain variable region (hVβ) and the mouse TCR β chain constant region (mCβ).

[0015] Figure 1B is a schematic diagram illustrating a method for selectively expanding a plurality of T cells expressing mTCR according to an embodiment of the present invention.

[0016] Figure 2 Experimental data (dot plots) are shown, illustrating (i) CD3, CD4 or CD8 expression and (ii) mTCRβ expression detected by FACS in anti-mutated ERBB2mTCR or anti-mutated ERBB2IP mTCR-electroporated cells (ERBB2mutTCR or ERBB2IPmutTCR) subjected to a 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) were used as negative controls. The numbers in the dot plots are mTCRβ expression detected. + The percentage of cells.

[0017] Figure 3A and 3B is shown to resist the mutant ERBB2IP mTCRβ + (ERBB2IPmutTCR) (A) or anti-mutated ERBB2 (ERBB2mutTCR) mTCRβ + (B) Graph showing the percentage (%) of cells that also expressed CD3, CD4, or CD8 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 subjected to standard REP with OKT3 Ab instead of selective expansion with H57 Ab served as a positive control for nonspecific T cell growth.

[0018] Figure 4Ais a schematic diagram illustrating a method for selectively expanding a plurality of T cells expressing a TCR comprising a murine constant region (mTCR) according to an embodiment of the present invention.

[0019] Figure 4B Schematic diagram illustrating the binding of H57 Ab to the murine TCRβ chain constant region (mCβ) of TCR. The other components of TCR include the human TCRα chain variable region (hVα), the murine TCRα chain constant region (mCα), a synthetic linker sequence (L), and the human TCRβ chain variable region (hVβ).

[0020] Figures 5A-5D Experimental data (dot plots) illustrating CD3 expression and murine TCRβ 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 SBTS plasmid encoding mTCR (4149-TCRa2b2 / pSBSO) and SBTS plasmid encoding transposase described in Example 3 (pKan-CMV-SB11) (D) are shown. Numbers in the dot plots are CD3 + / mTCRβ + (upper right quadrant), CD3 + / mTCRβ - (lower right quadrant), CD3 - / mTCRβ - (lower left quadrant) and CD3 - / mTCRβ + Percentage of cells (upper left quadrant).

[0021] Figure 6 Experimental data (dot plots) are shown, illustrating mTCR-electroporated cells (mTCR in REP) undergoing selective amplification with indicated concentrations of H57 Ab (ng / mL) at indicated starting numbers of electroporated cells. + CD3 expression and mTCRβ expression detected by FACS in 100 μM WT cells. Cells electroporated with electroporation buffer (mock) only (no TCR) were used as negative controls. The numbers in the dot plots are CD3 + / mTCRβ + (upper right quadrant), CD3 + / mTCRβ - (lower right quadrant), CD3 - / mTCRβ - (lower left quadrant) and CD3 - / mTCRβ + Percentage of cells (upper left quadrant).

[0022] Figure 7 Experimental data (dot plots) are shown, illustrating CD3 expression and mTCRβ expression detected by FACS in mTCR-electroporated cells that underwent secondary expansion with H57 Ab or OKT3 Ab and were stained or unstained. Experimental data (dot plots) are also shown, illustrating CD4 and CD8 expression detected by FACS in mTCR-electroporated cells that underwent secondary expansion with OKT3 Ab.

[0023] Fig. 8A and 8B Experimental data (dot plots) are shown, illustrating the untransduced (UT) ( Figure 8B ) and transduced ( Fig. 8A ) cells for CD8 expression and anti-MART-1 TCR expression.

[0024] Figure 8C Experimental data (dot plots) are shown, illustrating the presence of mTCRb+ cells before expansion (pre-REP) and after dilution to approximately 5% by FACS. Fig. 8A CD8 expression and anti-MART-1 TCR expression of transduced cells.

[0025] Fig. 9 Experimental data (dot plots) are shown, illustrating the expression of UT cells detected by FACS after expansion with (i) 50 CU or 500 CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"). Figure 8C CD8 expression and anti-MART-1 TCR expression of diluted transduced cells.

[0026] Fig. 10A Figure 2 shows the expression of UT cells or TCRb detected after amplification with (i) 50 CU or 500 CU of IL-2 and (ii) OKT3 Ab or H57Ab ("mTCRb"). Figure 8C Figure 3. Graph of the percentage of transduced cells for dilutions of transduced cells.

[0027] Fig. 10B Figure 2 shows the expression of UT cells or H57 Ab ("mTCRb") after expansion with (i) 50CU or 500CU IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"). Figure 8C A graph of the fold expansion achieved for the dilutions of transduced cells.

[0028] Figures 11A-11C Experimental data (dot plots) are shown, illustrating that in UT cells ( Fig.11A ) or before four-fold dilution ( Fig. 11B ) and after ( Fig. 11C) CD8 expression and mTCRb expression detected by FACS before expansion of cells transduced with anti-MART-1 TCR.

[0029] Figures 12A-12E Experimental data (dot plots) are shown, illustrating the difference between the treatment with OKT3 and 500CU IL-2 ( Fig. 12A )、H57(mTCRb) and 500CU IL-2( Fig. 12B )、H57(mTCRb) and 50CU IL-2( Fig. 12C ), or H57(mTCRb) and no IL-2( Fig.12D ) Expand UT cells ( Fig.12E )or Fig. 11C CD8 expression and mTCRb expression were then detected by FACS after dilution of TCR-transduced cells.

[0030] Fig.13A The results show that UT cells or Fig. 11C Figure 3. Graph of the percentage of transduced cells for dilutions of transduced cells.

[0031] Fig. 13B Figure 2 shows that after expansion with (i) 0CU, 50CU or 500CU of IL-2 and (ii) OKT3 Ab or H57 Ab ("mTCRb"), UT cells or Fig. 11C A graph of the fold expansion achieved for the dilutions of transduced cells.

[0032] Figures 14A-14F Shown is the description before amplification ( Fig.14A ) or with OKT3 (30 ng / ml) ( Fig. 14B ) or H57(mTCRb)(5ng / ml( Fig.14F )、10ng / ml( Fig.14E )、50ng / ml( Fig.14D ) or 500ng / ml( Fig. 14C )) Experimental data of CD8 expression and mTCRb expression detected by FACS after expansion of transduced cells (dot graph). Fig.15 is a graph showing the fold expansion achieved for mTCR-transduced cells after amplification with OKT3 (30 ng / ml) or H57(mTCRb) (5, 10, 50 or 500 ng / ml).

[0033] Figures 16A-16EExperimental data (dot plots) are shown, illustrating CD8 expression and mTCRb expression detected by FACS after a second round of expansion of mTCR-transduced cells that underwent a first round of expansion with H57. The second round of expansion was performed with OKT3 (30 ng / ml) ( Fig.16A ) or H57(mTCRb)(5ng / ml( Fig.16E )、10ng / ml( Fig.16D )、50ng / ml( Fig. 16C ) or 500ng / ml( Fig. 16B ))conduct.

[0034] Fig.17 The graph shows the fold expansion of mTCR-transduced cells obtained after a second round of expansion of mTCR-transduced cells that underwent 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).

[0035] Fig.18 Experimental data (dot plots) are shown, illustrating CD3 expression and mTCRb expression detected by FACS 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 alone (mock) (no TCR) were used as negative controls.

[0036] Figures 19A-19B Experimental data (dot plots) are shown, illustrating CD3 expression and mTCRb expression by PBMCs from donors 1 and 2 transposed with 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2 and subjected to one round of expansion with H57 ( Fig.19B ). Unstained PBMC ( Fig.19A ) and PBMCs electroporated with electroporation buffer only (mock) (no TCR) ( Fig.19B ) was used as negative control.

[0037] Figures 20A-20B Shown are experimental data (dot plots) illustrating the generation of TCRs from cells transposed with 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2 and subjected to one round of amplification with H57 ( Fig. 20B ) CD3 expression and mTCRb expression of PBMCs from donors 1 and 2 that were then expanded using standard REP. Unstained PBMCs ( Fig. 20A ) and PBMCs electroporated with electroporation buffer only (mock) (no TCR) ( Fig. 20B) was used as negative control.

[0038] Fig.21A is a graph showing the concentration of IFN-γ secreted after co-culturing (i) DCs pulsed with DMSO, WT HUWE1 peptide or mutated (mut) HUWE1 peptide with (ii) cells from donor 1 transposed with 4149-HUWE1-TCR1. DCs cultured alone served as controls. Mean ± SEM; n = 3 technical replicates.

[0039] Fig. 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 transposed with 4149-TP53-TCRa2b2. DCs cultured alone served as controls. Mean ± SEM; n = 3 technical replicates.

[0040] Fig. 21C is a graph showing the concentration of secreted IFN-γ after co-culturing (i) DCs pulsed with DMSO, WT HUWE1 peptide, mut HUWE1 peptide and (ii) cells from donor 2 transposed with 4149-HUWE1-TCR1. DCs cultured alone served as controls. Mean ± SEM; n = 3 technical replicates.

[0041] Fig.21D is a graph showing the concentration of co-secreted IFN-γ after co-culture of (i) DCs pulsed with DMSO, WT TP53 peptide, or mut TP53 peptide with (ii) cells from donor 2 transposed with 4149-TP53-TCRa2b2. DCs cultured alone served as controls. Mean ± SEM; n = 3 technical replicates.

[0042] Fig. 22 is a schematic diagram illustrating a method for selectively expanding the number of T cells expressing a TCR comprising a murine constant region (mTCR) according to one embodiment of the present invention.

[0043] Fig.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 H57 selective amplification (striped bars), after enrichment with H57-conjugated beads (grey bars), and after standard REP with OKT3 (black bars). Cells were electroporated with 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2. Triplicate cuvettes were moved forward in parallel so that the data shown are mean values ​​+ / - SEM (n=3).

[0044] Fig. 23BIs 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-conjugated beads (grey bars), and after standard REP with OKT3 (black bars). Cells were electroporated with 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2. Triplicate cuvettes were moved forward in parallel so that the data shown are mean values ​​+ / - SEM (n=3).

[0045] Figures 23C-23D is a graph showing 4149-HUWE1-TCR1-transposition (mTCR+) measured on day 28 as described in Example 14 ( Fig.23C ) or 4149-TP53-TCRa2b2-transposase (mTCR+) ( Fig.23D ) . PBMCs electroporated with electroporation buffer alone (mock) (no TCR) were used as negative controls. Mock is represented by (1) and TCR-transposed cells are represented by (2). Triplicate cuvettes were moved forward in parallel so that the data shown are mean + / - SEM (n=3).

[0046] Fig.23E Is a graph showing the percentage of CD4+mTCRβ+ cells (open bars) or CD8+mTCRβ+ cells (solid bars) measured on day 28 (after OKT3 REP) of the method described in Example 14. TCR is 4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2. Triplicate cuvettes were moved forward in parallel so that the data shown are mean values ​​+ / - SEM (n=3).

[0047] Fig.24A are graphs showing the percentage of cells positive for the indicated markers measured before (day 1) (open bars) or after (day 28) expansion of cell numbers (filled bars). Cells were transposed with 4149-HUWE1-TCR1.

[0048] Fig. 24B Graph showing the percentage of mTCRβ+ cells with the indicated phenotype measured before (day 1) (open bars) or after (day 28) (filled bars) expansion of cell numbers. Cells were transposed with 4149-HUWE1-TCR1. The phenotype was central memory T (T CM ) cells, memory stem T cells (T SCM cells), natural T cells (T N ), effector memory T cells (T EM ) and effector memory RA T cells (TEMRA ).

[0049] Fig.24C are graphs showing the percentage of cells positive for the indicated markers measured before (day 1) (open bars) or after (day 28) (filled bars) expansion of cell numbers. Cells were transposed with 4149-TP53-TCRa2b2.

[0050] Fig.24D Graphs showing the percentage of mTCRβ+ cells with the indicated phenotype measured before (day 1) (open bars) or after (day 28) (filled bars) expansion of cell numbers. Cells were transposed with 4149-TP53-TCRa2b2.

[0051] Fig.25A is a graph showing the concentration of IFN-γ secreted after co-culture of (i) DCs pulsed with DMSO or 10, 1 or 0.1 μg / mL of WT HUWE1 peptide (open bars) or mutHUWE1 peptide (filled bars) with cells transposed with 4149-HUWE1-TCR1.

[0052] Fig.25B is a graph showing the concentration of secreted IFN-γ after co-culture of (i) DCs pulsed with DMSO or 10, 1 or 0.1 μg / mL of WT TP53 peptide (open bars) or mutTP53 peptide (solid bars) with (ii) cells transposed with 4149-TP53-TCRa2b2. DETAILED DESCRIPTION OF THE INVENTION

[0054] Embodiments of the present invention provide methods for selectively amplifying T cell numbers. The method may include modifying human T cells to express TCR, wherein TCR includes a mouse constant region (hereinafter referred to as "mTCR"). The method of the present invention may provide any one or more of a variety of advantages. For example, the method of the present invention may provide a selective amplification of the number of T cells expressing mTCR to exceed the number of cells that do not express mTCR. As described herein, compared with a cell group prepared by a method for non-selectively amplifying T cell numbers, the method of the present invention can provide a greater proportion of cells expressing mTCR for a cell group. Not subject to the constraints of a particular theory or mechanism, it is believed that compared with a cell group with a smaller proportion of cells expressing mTCR, a cell group with a greater proportion of cells expressing mTCR can provide one or both of the target cancer cell destruction and improved cancer treatments that are improved.

[0055] TCR generally comprises two polypeptides (i.e., polypeptide chains), such as the α-chain of TCR, the β-chain of TCR, the γ-chain of TCR, the δ-chain of TCR or a combination thereof. Such polypeptide chains of TCR are known in the art. mTCR can comprise any amino acid sequence, as long as the mTCR comprises a mouse constant region and can specifically bind to and immunorecognize an antigen, such as an antigen or an epitope thereof associated with a disease.

[0056] mTCR can be an exogenous TCR, i.e., a TCR that is not natural to T cells (not naturally present on T cells). Exogenous TCR can be a recombinant TCR. Recombinant TCR is a TCR generated by recombinant expression of one or more exogenous TCR α-, β-, γ- and / or δ-chain encoding genes. Methods for preparing recombinant TCRs are known in the art.

[0057] In one embodiment of the present invention, the mTCR comprises two polypeptide chains, each of which comprises a variable region comprising a complementarity determining region (CDR) 1, CDR2 and CDR3 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.

[0058] In one embodiment, the mTCR may comprise the amino acid sequence of the variable region of the TCR comprising the above CDRs. In this regard, the TCR may comprise an α chain variable region and a β chain variable region.

[0059] In one embodiment of the invention, mTCR also includes a mouse constant region in addition to the above-mentioned variable region or CDR. Preferably, mTCR includes both α chain mouse constant region and β chain mouse constant region. As used herein, the term "mouse" refers to any component of TCR or TCR described herein (e.g., complementary determining region (CDR), variable region, constant region, α chain and / or β chain), meaning a TCR (or its components) derived from a mouse, i.e., a TCR (or its components) derived from a mouse T cell or once expressed by a mouse T cell.

[0060] mTCR can include a TCR α chain containing a variable region and a constant region, and a TCR β chain containing a variable region and a constant region. The α chain variable region and the β chain variable region are collectively referred to as the "variable region" of TCR below. The α chain constant region and the β chain constant region are collectively referred to as the "constant region" of TCR below.

[0061] In one embodiment of the invention, mTCR is a mouse TCR. Mouse TCR may include a polypeptide chain completely derived from a mouse. In this regard, mouse TCR may include 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.

[0062] In another embodiment of the present invention, mTCR is a chimeric or hybrid TCR composed of an amino acid sequence derived from a TCR from two different mammalian species (i.e., mouse and non-mouse species). For example, mTCR may include a human variable region and a mouse constant region. Examples of chimeric TCRs comprising human variable regions and mouse constant regions are disclosed in patent application number 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.

[0063] In one embodiment of the invention, the mTCR is an antigen-specific TCR. As used herein, the phrases "antigen-specific" and "antigen specific" mean that the mTCR can specifically bind to and immunologically recognize an antigen or an epitope thereof, such that the binding of the mTCR to the antigen or an epitope thereof triggers an immune response.

[0064] The antigen recognized by the antigen-specific mTCR can be any antigen that is characteristic of the disease. For example, the antigen can be, but is not limited to, a cancer antigen (also referred to as 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.

[0065] As used herein, the term "cancer antigen" refers to any molecule (such as protein, polypeptide, peptide, lipid, carbohydrate, etc.) expressed or overexpressed by a tumor cell or cancer cell alone or mainly, so that the antigen is related to a tumor or cancer. Cancer antigens can be expressed by normal, non-tumor or non-cancerous cells in addition. However, in such cases, the expression of cancer antigens by normal, non-tumor or non-cancerous cells is not as robust as the expression of cancer antigens by tumors or cancer cells. In this regard, compared with the expression of antigens by normal, non-tumor or non-cancerous cells, tumors or cancer cells can overexpress antigens or express antigens at significantly higher levels. In addition, cancer antigens can be expressed by cells in different developmental or mature states in addition. For example, cancer antigens can be expressed by cells in embryonic or fetal stages in addition, and the cells are usually not present in adult hosts. Alternatively, cancer antigens can be expressed by stem cells or precursor cells in addition, and the cells are usually not present 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, mutated KRAS, NY-ESO-1 (also known as CAG-3), MAGE-1, MAGE-3, etc.

[0066] In one embodiment, the cancer antigen is a neoantigen. A neoantigen is a tumor-specific or cancer-specific antigen generated by a gene mutation that occurs in a tumor cell or cancer cell during the neoplastic transformation phase. The neoantigen may be unique to the patient. In a preferred embodiment, the neoantigen is an immunogenic neoantigen.

[0067] Cancer antigens can be antigens expressed by any cell of any cancer or tumor, including cancers and tumors described herein. Cancer antigens can be cancer antigens of only one type of cancer or tumor, such that the cancer antigen is associated with only one type of cancer or tumor or is a feature of only one type of cancer or tumor. Alternatively, cancer antigens can be cancer antigens of more than one type of cancer or tumor (e.g., can be characteristic). For example, cancer antigens can be expressed by both breast cancer cells and prostate cancer cells, and are not expressed at all by normal, non-tumor or non-cancerous cells.

[0068] The disorder associated with or characterized by an antigen recognized by an antigen-specific mTCR can be any disorder. For example, as discussed herein, the disorder can be a cancer or a viral disorder.

[0069] The cancer can be any cancer, including any of the following: acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, 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 urinary bladder cancer.

[0070] For the purposes of this document, "viral disorders" means disorders that can be transmitted from person to person or from organism to organism and are caused by viruses. In one embodiment of the invention, the viral disorder is caused by a virus selected from the group consisting of herpes viruses, poxviruses, hepadnaviruses, papillomaviruses, adenoviruses, coronaviruses, orthomyxoviruses, paramyxoviruses, flaviviruses, and caliciviruses. For example, the viral disorder can 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.

[0071] The viral condition 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.

[0072] In one embodiment of the invention, the method includes modifying human T cells to express mTCR. T cells can be isolated or purified. As used herein, the term "isolated" means that it has been removed from its natural environment. As used herein, the term "purified" means that the purity has been improved, wherein "purity" is a relative term and is not necessarily interpreted 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.).

[0073] The T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells obtained from non-mouse mammals. If obtained from non-mouse mammals, the T cells can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, spleen, or other tissues or body fluids. The cells can also be enriched or purified. Preferably, the T cells are human T cells. The T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, such as Th1 and Th2 cells, CD4 + T cells, CD8 + T cells (such as cytotoxic T cells), peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating cells (TIL), memory T cells, natural T cells, etc.

[0074] The method may include using any technology suitable for introducing mTCR or nucleic acid encoding mTCR into human T cells and obtaining human T cells to modify human T cells to express mTCR. Such technology is described in, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Examples of technologies that can be used to modify human T cells to express TCR include, but are not limited to, transfection, conversion, transduction, electroporation, and gene editing techniques. In one embodiment 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, Sleeping Beauty transposon system (SBTS) (available from Intrexon (Germantown, MD) and Ziopharm (Boston, MA)), PIGGYBAC transposon system (available from Transposagen, Lexington, KY) and Tol2 transposon system. Examples of gene editing technologies may 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).

[0075] The method can also include producing a cell population comprising a plurality of human T cells expressing mTCR and a plurality of human T cells not expressing mTCR. Modifying human T cells to express mTCR can be performed with variable efficiency. Therefore, modifying human T cells to express mTCR can produce a mixed cell population, which includes cells expressing mTCR and cells not expressing mTCR.

[0076] The method can also include culturing the 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, wherein the antibody specifically binds to the murine constant region of the mTCR, thereby selectively expanding the number of T cells that express the mTCR over the number of T cells that do not express the mTCR (also referred to herein as "selective expansion").

[0077] The irradiated feeder cells may include any irradiated feeder cells suitable for expanding the number of T cells. In embodiments 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 employed is not limited and may be selected by the skilled artisan based on a variety of factors, such as, for example, the application and the desired number of cells to be obtained. For example, a plurality of 2 x 10 7 Multiple 1x 10 8 Irradiated feeder cells can be used for medium-scale expansion. About 2 to about 30 times the 5x 10 8 Irradiated feeder cells can be used for large-scale expansion (e.g., clinical production) (typically up to about 1.5 x 10 10 ). For example, the method may use about 1x 10 9 About 4x 10 9 Allogeneic feeder cells and / or autologous feeder cells, preferably about 2 x 10 9 About 3x 10 9 Allogeneic feeder cells and / or autologous feeder cells.

[0078] 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.

[0079] The method may also 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 of any isotype, such as 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, such as an antibody isolated and / or purified from mammals such as rabbits, goats, horses, chickens, hamsters, humans, etc. Alternatively, the antibody may be a genetically engineered antibody, such as a humanized antibody or a chimeric antibody. The antibody may be in monomeric or polymeric form. In addition, the antibody or its antigen binding fragment may have any level of affinity or avidity to the mouse constant region of the mTCR.

[0080] In one embodiment of the invention, the antibody comprises two polypeptide chains (heavy chain and light chain), each of which comprises a variable region comprising complementary determining regions (CDR) 1, CDR2 and CDR3 of the antibody. The antibody may comprise a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3. In one embodiment of the invention, the antibody comprises a heavy chain variable region and a light chain variable region.

[0081] The antigen binding portion of an antibody can be any portion having at least one antigen binding site. In one embodiment of the invention, the antigen binding portion may include 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 invention, the antigen binding portion may include 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), F(ab')2 fragment, Fab' fragment, Fv fragment, single chain variable region fragment (scFv), disulfide bond-stabilized variable region fragment (dsFv), scFv2CH3, scFv4, scFv3, scFv2, scFv-Fc or (scFv)2. Conventional recombinant DNA process technology can be used to generate a single chain variable region fragment (scFv), which is a fusion protein comprising a variable (V) domain of an antibody heavy chain connected to the V domain of an antibody light chain via a synthetic peptide. Similarly, disulfide bond-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 portions. Unless otherwise specified, antibodies and antigen binding portions thereof are hereinafter collectively referred to as "antibodies".

[0082] The antibody can be any antibody that specifically binds to the mouse constant region of mTCR. In one embodiment of the invention, the antibody specifically binds to the mouse constant region of mTCR and does not bind to any part of human TCR, such as any part of human TCR constant region or human TCR complex. The TCR complex comprises TCR α and β chains with three dimer signaling chains: CD3 δ / ε, CD3 γ / ε and CD247 ζ / ζ or ζ / η. CD3 δ / ε and CD3 γ / ε chains are collectively referred to as CD3 complexes.

[0083] The antibody can specifically bind to the mouse constant region of the α chain of mTCR or the mouse constant region of the β chain of mTCR. In a preferred embodiment, the antibody specifically binds to the mouse constant region of the β chain of mTCR. The mouse constant region of the mTCR β chain to which the antibody specifically binds may include the following or be composed of the following: SEQ ID NO:1 amino acid sequence (amino acid sequence of the full-length mouse constant region of the mTCR β chain). The antibody can specifically bind to any part of the mouse constant region of the mTCR β chain. For example, the antibody can specifically bind to SEQ ID NO:2 amino acid sequence (amino acid sequence of an exemplary minimum epitope of the full-length mouse constant region of the mTCR β chain). In one embodiment of the invention, the antibody specifically binds to the 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.

[0084] Antibodies that specifically bind to the mouse constant region of mTCR are commercially available. For example, the antibody that specifically binds to the mouse constant region of the β chain of mTCR is H57 antibody (also referred to as H57-597) (available from Biolegend, San Diego, CA). H57 is an American hamster IgG antibody and is described in, for example, Kubo et al., J.Immunol., 142 (8): 2736-42 (1989) and Grégoire et al., PNAS, 88: 8077-81 (1991). The epitope of H57 antibody is described in, for example, Wang et al., EMBO J., 17 (1): 10-26 (1998). 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.

[0085] In one embodiment of the invention, the antibody comprises a heavy chain variable region and a light chain variable region. For example, the antibody (Ab) may comprise, consist of, or consist essentially of the following: the amino acid sequence of SEQ ID NO:3 (variable region of H57 Ab heavy chain) or the amino acid sequence of SEQ ID NO:4 (variable region of H57 Ab light chain) or both the amino acid sequences of SEQ ID NO:3 and 4. Preferably, the antibody comprises the amino acid sequences of both SEQ ID NO:3 and 4. In one embodiment of the invention, the antibody comprises complementary determining regions (CDR) 1, CDR2 and CDR3 of the H57 Ab heavy chain of SEQ ID NO:3, and CDR1, CDR2 and CDR3 of the H57 Ab light chain of SEQ ID NO:4.

[0086] In one embodiment of the invention, the antibody comprises a heavy chain and a light chain comprising the variable regions described above. For example, the antibody may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 5 (H57 Ab heavy chain) or the amino acid sequence of SEQ ID NO: 6 (H57 Ab light chain), or both the amino acid sequences of SEQ ID NO: 5 and 6. Preferably, the antibody comprises the amino acid sequences of both SEQ ID NO: 5 and 6.

[0087] As described herein, culturing a cell population in the presence of (i) irradiated feeder cells, (ii) one or more cytokines, and (iii) antibodies advantageously selectively expands the number of T cells expressing mTCR over the number of T cells not expressing mTCR. In this regard, the method of the present invention can advantageously provide a cell population having a greater proportion of cells expressing mTCR compared to a method of amplifying cell numbers without using antibodies that specifically bind to the murine constant region of TCR. In one embodiment of the invention, the method increases the number of T cells expressing mTCR by about 5-fold or less to about 4,000-fold or greater. For example, the methods of the 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 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 the range of any two of the aforementioned values. The aforementioned multiple expansion can be achieved over a time period of about 10 to about 14 days, preferably about 14 days. The aforementioned multiple expansion can be achieved over a time period of about 10 to about 14 days, preferably about 14 days. The multiple expansion achieved by the method of the present invention can be highly variable and can be donor-dependent.

[0088] In one embodiment of the invention, the method produces a cell population selected for amplification, wherein about 10% to about 95% of the cells in the selected amplified population express a TCR comprising a mouse constant region. In this regard, the method can produce a selectively amplified cell population, wherein 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 in the selectively amplified population express mTCR. The method can produce a selectively expanded cell population, wherein 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 within any two of the foregoing ranges, of the cells in the selectively expanded population express mTCR.

[0089] The methods of the present invention can advantageously produce any number of mTCR-expressing T cells suitable for any of a variety of applications. In one embodiment of the present invention, the method can produce about 1 x 10 6 About 1x 10 11 For example, in a small container (e.g., a T25 flask), the method of the present invention can produce about 1 x 10 6 About 1x 10 7 In a larger container (e.g., a T175 flask), the method of the present invention can produce about 5x10 6 About 3x10 7 In other containers (such as GREX flasks, available from Wilson Wolf Manufacturing, New Brighton, MN), the method of the present invention can produce about 1 x 10 9 About 1x 10 10 T cells expressing mTCR. Approximately 1x10 6 About 1x 10 10 A population of 10 T cells can be used for small-scale screening experiments. A larger number of cells expressing mTCR, such as about 1.5 x 10 10 Or more may also be obtained using the methods of the invention, such as for clinical applications. The number of mTCR-expressing T cells produced by the methods of the invention may be highly variable and may be donor-dependent.

[0090] The method may include performing no more than a single round of selective amplification of the number of mTCR-expressing cells or multiple rounds of selective amplification of the number of mTCR-expressing cells. In one embodiment of the invention, the method includes performing one or more rounds of selective amplification as described herein with respect to other aspects of the invention, followed by one or more rounds of non-selective amplification of the number of cells. In this regard, the method may also include culturing human T cells in the presence of (i) irradiated allogeneic feeder cells and irradiated autologous feeder cells, (ii) one or more cytokines, and (iii) antibodies or antigen binding portions thereof that specifically bind to human CD3 complexes (also referred to herein as "non-selective amplification"). Expansion of the number of T cells can be achieved by any of a variety 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, an antibody or antigen-binding portion thereof that specifically binds to a human CD3 complex in the presence of feeder lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15) can be used to non-selectively expand the number of T cells, with IL-2 being preferred. An example of an antibody that specifically binds to a human CD3 complex is OKT3 (available from Ortho-McNeil, Raritan, NJ).

[0091] 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 one embodiment of the invention, the method produces a cell population, wherein about 20% to about 99% of the cells in the population express a TCR comprising a mouse constant region. Multiple rounds of selective amplification, or one or more rounds of selective amplification followed by one or more rounds of non-selective amplification can produce a cell population, wherein 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 can produce a population of cells wherein 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 within any two of the foregoing ranges, of the cells in the population express an mTCR.

[0092] In one embodiment of the invention, the method also includes using an antibody or its antigen binding portion specifically bound to the mouse constant region of TCR to separate T cells expressing TCR from T cells not expressing TCR. In this regard, the method may include making a mixed cell population containing cells expressing mTCR and cells not expressing mTCR physically contact with an antibody so that the antibody specifically binds to the mouse constant region of TCR. The antibody may be attached to a support such as a bead. The method may also include washing antibodies and cells so that all or part of the cells not expressing mTCR are removed from cells expressing mTCR. The method may further include eluting mTCR-expressing cells from antibodies. Examples of techniques for separating T cells are described in, for example, Deniger et al., Mol. Ther., 24 (6): 1078-89 (2016) and Field et al., PLoS One, 8 (6): e68201 (2013).

[0093] The method of the present invention can advantageously provide a cell group enriched for expressing mTCR cells.Therefore, one embodiment of the present invention provides a cell group, which comprises the T cells of the selective amplification number prepared by any method as described herein.Cell group can be a heterologous group, which comprises mTCR-expressing cells together with at least one other cell, such as T cells not expressing mTCR, or cells other than T cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc.Alternately, cell group can be a substantially homogeneous colony, wherein the colony mainly includes mTCR-expressing T cells (such as, substantially composed of mTCR-expressing T cells).The colony can also be a clonal cell group, wherein all cells of the colony are clones of single T cells expressing mTCR, so that all cells of the colony express mTCR.In one embodiment of the present invention, cell group is a clonal colony of T cells comprising expressing mTCR.

[0094] The cell populations of the present invention may be isolated and / or purified. As used herein, the term "isolated" means having been removed from its natural environment. As used herein, the term "purified" means that the purity has been increased, wherein "purity" is a relative term and is not necessarily interpreted as absolute purity. For example, the purity may be at least about 50%, may be greater than about 60%, about 70%, about 80%, about 90%, about 95%, or may be about 100%.

[0095] The cell population of the present invention can be formulated into a composition, such as a pharmaceutical composition. In this regard, the present invention provides a pharmaceutical composition comprising any cell population described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can comprise any cell population of the present invention and another one or more pharmaceutically active agents or drugs (such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.) combination.

[0096] Preferably, the carrier is a pharmaceutically acceptable carrier. Regarding pharmaceutical compositions, the carrier can be any of those conventionally used for T cells. Methods for preparing the composition that can be administered are known or apparent to those skilled in the art, and are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press (2012). Preferably, under the conditions of use, the pharmaceutically acceptable carrier is a pharmaceutically acceptable carrier that does not have harmful side effects or toxicity.

[0097] The selection of carrier can be determined by the ad hoc method for administering cell population of the present invention. Therefore, there are multiple suitable preparations of pharmaceutical composition of the present invention. Suitable preparations can include any one of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral or intraperitoneal administration. More than one approach can be used to administer cell population of the present invention, and in some cases, a specific approach can provide a more direct and more effective response than another approach.

[0098] Preferably, the cell population of the present invention is administered intravenously by injection, for example. The pharmaceutically acceptable carrier for the injection cell may include any isotonic carrier, such as, for example, normal saline (about 0.90% w / v NaCl aqueous solution, about 300mOsm / L NaCl aqueous solution or about 9.0g NaCl / liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% glucose aqueous solution or Ringer's lactate. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin. The pharmaceutical composition for infusion may or may not contain IL-2. If the pharmaceutical composition contains IL-2, a concentration of about 300IU / mL may be used.

[0099] For purposes of the present invention, the amount or dosage (such as cell number) administered should be sufficient to achieve, for example, treatment or prevention response in a mammal within a reasonable time frame. For example, the dosage (such as, cell number) should be sufficient to bind to the antigen associated with the disorder, or detect, treat or prevent the disorder within a time period of about 2 hours or longer (such as 12 hours) to 24 or more hours from the time of administration. In certain embodiments, the time period can be even longer. The dosage will be determined by the efficacy of the cell mass and the condition of the mammal (such as a person) and the body weight of the mammal (such as a person) to be treated.

[0100] Many assays for determining the administered dose are known in the art. For the purposes of the present invention, assays can be used to determine the starting dose to be administered to a mammal, comprising administering a given dose of T cells to a group of mammals (each of which is administered a different dose of T cells) and then comparing the extent of target cell lysis or IFN-γ secretion by such T cells expressing mTCR. After administering a certain dose, the extent of target cell lysis or IFN-γ secretion can be determined by methods known in the art.

[0101] The dosage of the cell population of the invention will also be determined by the presence, 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 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 invention to be administered, the route of administration, and the severity of the condition being treated. In one embodiment, the number of cells administered per infusion may vary, e.g., from about 1 x 10 6 About 1x 10 12 In some embodiments, at least about 1.5 x 10 10 cells.

[0102] The cell populations produced by the methods of the invention can be used to treat or prevent disorders, such as cancer. Therefore, another embodiment of the invention provides a method for treating or preventing a disorder in a mammal, the method comprising selectively expanding the number of T cells according to any method described herein for other aspects of the invention, and administering the selectively expanded number of T cells to the mammal in an amount effective to treat or prevent the disorder in the mammal.

[0103] For the purposes of the methods of the invention, wherein a cell population is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0104] In one embodiment of the invention, the disorder is cancer.The cancer may be any cancer described herein with respect to the other aspects of the invention.

[0105] In one embodiment of the invention, the disorder is a viral disorder.The viral disorder may be any viral disorder as described herein in relation to other aspects of the invention.

[0106] 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 Felidae (cats) and Canidae (dogs). More preferably, the mammal is from the order Artiodactyla, including Bovidae (cows) and Suidae (pigs), or belongs to the order Persistodactyla, including Equine (horses). Most preferably, the mammal belongs to the order Primates, Ceboids, or Simians (monkeys) or to the order Anthropoids (humans and apes). A particularly preferred mammal is a human.

[0107] 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 varying degrees of treatment or prevention, and those of ordinary skill in the art recognize that they have potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide any level of cancer treatment or prevention in any number of mammals. In addition, the treatment or prevention provided by the methods of the present invention can include the treatment or prevention of one or more conditions or symptoms of conditions being treated or prevented, such as cancer. For example, the treatment or prevention provided by the methods of the present invention can include promoting the regression of tumors. Likewise, for purposes herein, "prevention" can encompass delaying the onset of a condition or its symptoms or illness.

[0108] The following examples further illustrate the invention but, of course, should not be construed as limiting its scope in any way.

[0109] Example 1

[0110] This example demonstrates that selectively expanding the number of T cells expressing a TCR comprising a murine constant region (mTCR) increases the number of cells expressing the mTCR.

[0111] RNA encoding two TCRs (one with antigenic specificity for cancer-specific, mutated ERBB2 neoantigens and the other with antigenic specificity for cancer-specific, mutated ERBB2IP neoantigens) was isolated from tumor infiltrating lymphocytes (TILs) isolated from cancer patients. cDNA was amplified from RNA by reverse transcription, and then cDNA copies were amplified by PCR. mTCR constructs (one for each TCR) were prepared as described in Deniger et al., Mol. Ther., 24(6): 1078-89 (2016). In short, 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 located 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)) and described in Deniger et al., PLoS One, 10(6):e0128151 (2015) and Singh et al., Cancer Res., 71(10):3516-27 (2011).

[0112] Electroporation and selective expansion of peripheral blood mononuclear cells (PBMCs) with mTCR according to one embodiment of the present invention are illustrated. + A schematic diagram of the method for counting T cells is shown in Figure 1B In. Figure 1B SBTS plasmids encoding each of the two mTCRs and SBTS plasmid encoding the transposase (5 μg) were electroporated into autologous PBMCs (2×10 7 24 hours later, a mixed electroporated cell population containing cells expressing mTCR and cells not expressing mTCR was obtained.

[0113] like Figure 1B As shown, T cells expressing the murine TCR constant region (mTCR + In order to selectively amplify mTCR + T cells were isolated from mixed electroporated cell populations (2 x 10 6) and irradiated peripheral blood lymphocytes (PBL) (2 x 10 7 ) co-cultivation.

[0114] Selective amplification of mTCR + After counting 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 only (mock) (no TCR) were used as negative controls.

[0115] The stained cells were counted by fluorescence activated cell sorting (FACS). The results are shown in Figure 2 In. Figure 2 As shown, selective amplification with H57 Ab increased anti-mutated ERBB2 mTCR compared with OKT3 + Number of T cells and anti-mutated ERBB2IP mTCR + The number of T cells.

[0116] Example 2

[0117] This example shows that a higher number of mTCRs were obtained with a higher concentration of H57 Ab. + .

[0118] PBMCs were electroporated with anti-ERBB2IP mTCR or anti-ERBB2 mTCR constructs as described in Example 1. As described in Example 1, 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) were used to selectively expand T cells expressing the murine TCR constant region (mTCR + The number of T cells).

[0119] In mTCR + After selective expansion of the number of T cells, cells were stained with (i) anti-CD3 Ab, anti-CD4 Ab or anti-CD8 Ab and (ii) anti-mTCRβ antibody (H57). PBMC (mock) electroporated with electroporation buffer alone (no TCR) was used as a negative control. Electroporated cells subjected to standard REP with OKT3 Ab (30 ng / mL) instead of selective expansion with H57 Ab were used as a positive control for nonspecific T cell growth.

[0120] The stained cells were counted by FACS. The results are shown in Figure 3A and 3B In. Figure 3A and3B As shown in Figure 2, higher numbers of anti-mutated ERBB2 mTCRs were obtained with higher concentrations of H57Ab. + and anti-mutated ERBB2IP mTCR + cell.

[0121] Example 3

[0122] This example demonstrates a method for selectively expanding the number of T cells expressing mTCR.

[0123] RNA encoding a TCR with antigenic 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, followed by PCR amplification of the cDNA copies. mTCR constructs were prepared by fusion of 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 ). The mTCR construct was synthesized and cloned into the SBTS plasmid.

[0124] Example Electroporation of PBMCs with mTCR and Selective Expansion of mTCR According to One Embodiment of the Invention + A schematic diagram of the method for counting T cells is shown in Figure 4A In. Figure 4A As shown, autologous PBMCs were obtained from patients and cryopreserved. Cryopreserved PBMCs (200 g) were thawed at 20-23°C for 10 minutes. PBMCs were placed in 50 / 50 medium (3 x 10 6 The SBTS plasmid encoding mTCR and the SBTS plasmid encoding transposase are electroporated to autologous PBMC using AMAXA NUCLEOFECTOR II equipment and test kit (Lonza, Basel, Switzerland). Human T cell NUCLEOFECTOR solution (300 μL) contains the SBTS plasmid encoding mTCR (45 μg) and the SBTS plasmid encoding transposase (15 μg). The human T cell NUCLEOFECTOR solution is added to a cuvette (100 μL per cuvette). The electroporated cells are cultured overnight at 37 ° C in holes (5 mL 50 / 50 culture medium / well). After more than 18 hours, 50 U / mL broad-spectrum nuclease (benzonase) is incubated at 37 ° C for 1 hour. The cell colony of the electroporation mixed comprising the cell expressing mTCR and the cell not expressing mTCR is obtained.

[0125] like Figure 4A As shown, after culturing at 37°C for more than 18 hours, T cells expressing the mouse TCR constant region (mTCR + In order to selectively amplify mTCR + T cells were 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 (5x10 5 ) and irradiated peripheral blood lymphocytes (PBL) (1x 10 8 ) co-culture.

[0126] In selective amplification of mTCR + After the number of T cells was determined, mTCR was further expanded using a standard rapid expansion protocol (REP) + In standard REP, the number of selectively amplified mTCR + T cells (5x 10 6 ) and irradiated PBL (5x 10 8 ), OKT3 antibody (30 ng / mL) and IL-2 (3000 IU / mL). OKT3 antibody reacts with an epitope on the ε-subunit in the human CD3 complex.

[0127] Comparative Example 1

[0128] This example demonstrates the number of electroporated cells expressing mTCR in the absence of selective expansion.

[0129] As described in Example 4, autologous PBMCs were electroporated with SBTS plasmids encoding mTCR and SBTS plasmids encoding transposase. Negative controls included untransfected autologous PBMCs and PBMCs electroporated with electroporation buffer alone (mock). Electroporated cells were stained with anti-CD3 antibody and anti-mTCRβ antibody (H57). Unstained, untransfected autologous PBMCs were used as yet another negative control.

[0130] On the day after electroporation, stained cells were counted by FACS without selective expansion as described in Example 4. (neg) Set gate on cells. PI is a stain for dead cells. (neg) Cells are living cells.

[0131] The results are shown in Figures 5A-5D In. Figures 5A-5DAs shown, a modest number of cells in the total population (approximately 5% of total cells) expressed mTCR after electroporation without undergoing selective expansion.

[0132] Example 4

[0133] 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.

[0134] Autologous PBMCs were electroporated with SBTS plasmids encoding mTCR and transposase as described in Example 3. As described in Example 3, various starting numbers (1 x 10 5 , 5x 10 5 or 10x 10 5 ) of mTCR+ T cells. The experiment normalized the input to 5x 10 based on the initial frequency. 5 For example, 5% mTCR expression means that 1x 10 7 Total cells were entered into the expansion protocol to achieve 5 x 10 5 mTCR+T cells. PBMCs electroporated with electroporation buffer only (mock) were used as negative controls. Electroporated, selectively expanded cells were stained with anti-CD3 antibody and anti-mTCRβ antibody (H57).

[0135] Count the stained cells by FACS. (neg) The results are shown in Figure 6 and Table 1-2. Figure 6 As shown in Table 1-2, selective amplification with H57 Ab increased the number of mTCR + The number of T cells.

[0136] Table 1

[0137]

[0138] Table 2

[0139]

[0140] Example 5

[0141] This example shows that after the first amplification with H57 Ab, the second amplification with OKT3 Ab further increased mTCR + The number of cells.

[0142] As described in Example 4, (i) the initial number of 5 x 10 5 Electroporated cells selectively expanded with (ii) 250 ng / mL H57 Ab (referred to as "first expansion" in this example) at 400 mTCR+ T cells were further expanded in GREX flasks (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 "second expansion" in this example).

[0143] After the second expansion, the cells were stained with anti-CD3 antibody and anti-mTCRβ antibody (H57). The stained cells were counted by FACS. The FACS was gated on lymphocytes / PI(neg) cells. The results are shown in Figure 7 middle.

[0144] like Figure 7 As shown, after the first expansion using H57 Ab, the second expansion using OKT3 Ab further increased the number of mTCR+ cells. Compared with the second expansion using H57 Ab, the second expansion using OKT3 Ab increased the number of mTCR+ cells to a greater extent.

[0145] The percentage of viable cells, the total number of cells, the fold change in the number of cells achieved by the second expansion, the number of mTCR+ cells, and the fold change in mTCR+ cells achieved by the second expansion were also measured in cells undergoing the second expansion with OKT3 Ab and H57 Ab, and compared. The results are shown in Table 3. As shown in Table 3, after the first expansion with H57 Ab, the second expansion with OKT3 Ab further increased the mTCR + The second expansion using OKT3 Ab increased mTCR to a greater extent than the second expansion using H57 Ab. + The number of cells.

[0146] Table 3

[0147]

[0148] Example 6

[0149] This example demonstrates that selectively expanding the number of T cells expressing the mTCR increases the number of cells expressing the mTCR.

[0150] PBL were transduced with a γ-retroviral vector encoding murine F5 (mF5) anti-MART-1 TCR using standard methods on day 0. Transduced cells were prepared for a second expansion on day 10. Expansion was performed using (i) varying concentrations of IL-2, (ii) varying ratios of feeder cells, and (iii) OKT3 Ab (30 ng / ml) or H57 Ab (50 ng / ml) as shown in Table 4.

[0151] Table 4

[0152]

[0153] Figures 8A-8B The FACS graphs in the Figures show the percentage of untransduced and transduced cells expressing CD8 and mouse F5 (mF5) anti-MART-1 TCR before amplification with OKT3 Ab or H57 Ab (before rapid expansion protocol (REP)). The transduction efficiency before amplification was 83.9%. Prior to amplification, the transduced population was diluted to approximately 5% mouse TCRβ chain positive (mTCRb+) cells ( Figure 8C ).

[0154] (i) OKT3 (50 ng / ml) or H57 (50 ng / ml) in (ii) 500 or 50 CU / IL-2 Figure 8C The results after expansion of the cell number from the 5% mTCRb+ starting population are shown in Fig. 9 and 10A -10B. The number of cells was expanded by about 1500–2300-fold in the case of OKT3 but did not show a selective expansion of the number of cells in the mTCRb+ cell population. Alternatively, the number of cells was expanded less (500-800-fold) in the case of H57, however, they showed a 4-8-fold increase in the percentage of mTCRb+ cells. In short, using H57 in amplification produces more mTCRb+ cells.

[0155] Example 7

[0156] This example demonstrates that selectively expanding the number of T cells expressing the mTCR increases the number of cells expressing the mTCR.

[0157] PBL 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 prior to expansion with H57 or OKT3 (starting cell population) is shown in Figures 11A-11C The starting mTCR+ population initially had 64.7% mTCRb+ cells ( Fig. 11B The starting mTCR+ population was diluted four-fold to 14% mTCRb+ ( Fig. 11C).

[0158] The number of transduced and UT cells was expanded using (ii) OKT3 Ab or H57 Ab in the absence of IL-2, 50 CU IL-2, or 500 CU IL-2. The results are shown in Figures 12A-12E and 13A-13B.

[0159] After expansion, the OKT3 population did not show selective expansion of the entire mTCRb+ cell population, with approximately equal percentages of CD8+ and CD8-converted cells (5%). In contrast, both H57 expansion populations (500 or 50CU / IL-2, respectively) showed selective mTCRb+ expansion, 31.2% and 59.1%, respectively. H57 expansion using 50CU / IL-2 showed a nearly 2-fold increase in mTCRb+ cell expansion compared to cells grown in 500CU / IL-2.

[0160] As shown in Figures 13-13B, in the presence of 500 CU / IL-2, the number of cells expanded with OKT3 or H57 expanded by about 1400-1600 fold. However, H57 expansion with 50 CU / IL-2 expanded by about 3500-fold, more than twice that of the other tested conditions.

[0161] Example 8

[0162] This example demonstrates that selectively expanding the number of T cells expressing the mTCR increases the number of cells expressing the mTCR.

[0163] PBL 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 prior to expansion with H57 or OKT3 (pre-REP) is shown in Figures 14A-14F middle.

[0164] 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 Fig.15 In. Figures 14A-14F and Fig.15 As shown, the concentration of H57 antibody can be titrated down 10-fold without any adverse effect on selective mTCRb+ expansion. Likewise, the overall expansion fold was similar between all tested groups. Preferential expansion of CD8- cells after expansion with H57 was also observed.

[0165] Example 9

[0166] This example demonstrates that a second round of expansion with OKT3 or H57 further expands the number of cells that underwent the first round of H57 expansion in Example 8.

[0167] The mTCR-transduced cells that had undergone amplification with H57 Ab in Example 8 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.

[0168] The results are shown in Figures 16A-16E and Fig.17 The second expansion with OKT3 did not further enrich for mTCRb+ cells, but the number of cells did expand by an additional 1000-fold. The second expansion with H57 did not increase any additional enrichment of mTCRb+ cells and resulted in a slight decrease in mTCRb+ cells at 500 and 50 ng / ml. The expansion folds of the populations that underwent the second expansion with H57 were low, ranging from 200-500 fold.

[0169] Example 10

[0170] This example demonstrates expression of a mutant-specific TCR from a transposon the day after electroporation.

[0171] like Figure 4A As shown, autologous PBMCs were obtained and cryopreserved from healthy donor 1 and healthy donor 2. The cryopreserved PBMCs were thawed and spun (200 g) at 20-23°C for 10 minutes. The PBMCs were placed in 50 / 50 medium (3 x 10 6Cells / mL) and cultured at 37 ° C for 2 hours. Using AMAXA NUCLEOFECTOR II equipment and kit (Lonza, Basel, Switzerland), the SBTS plasmid encoding one of the two mTCRs and the SBTS plasmid encoding the transposase were electroporated into autologous PBMC. These two mTCRs are (1) 4149-HUWE1-TCR1 (Class I) and (2) 4149-TP53-TCRa2b2 (Class II). Human T cell NUCLEOFECTOR solution (300 μL) contains SBTS plasmids (45 μg) encoding mTCR and SBTS plasmids (15 μg) encoding transposase. After a 2-hour rest period, non-adherent PBMCs were collected by rotating (200 g) for 10 minutes at 20-23 ° C, the culture medium was removed and replaced with human T cell NUCLEOFECTOR solution containing SBTS plasmids (100 μL was added to each cuvette). The electroporated cells were cultured overnight at 37°C in wells (5 mL 50 / 50 medium / well). After >18 hours, 50 U / mL broad-spectrum nuclease was incubated for 1 hour at 37°C. A mixed electroporated cell population containing cells expressing mTCR and cells not expressing mTCR was obtained.

[0172] The day after electroporation, electroporated cells were stained with (i) anti-CD3 Ab and (ii) anti-mTCRβAb. Unstained PBMC, untransfected PBMC and PBMC electroporated with electroporation buffer (mock) only (no TCR) were used as negative controls. The expression of CD3 and mTCR by electroporated cells was measured by FACS (gating: lymphocytes\live (PI-)). The results are shown in Fig.18 In. Fig.18 As shown, the mutant-specific TCR was expressed from the transposon the day after electroporation.

[0173] Embodiment 11

[0174] This example demonstrates that one round of expansion with H57 results in a selective overgrowth of the number of mTCR+ T cells.

[0175] PBMCs from donors 1 and 2 were electroporated as described in Example 10. Figure 4A As shown, after culturing at 37°C for more than 18 hours, T cells expressing the mouse TCR constant region (mTCR + In order to selectively amplify mTCR +The number of T cells was determined by incubation with mTCR 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). + T cells (5x 10 5 ) and irradiated PBL (1x 10 8 ) co-culture.

[0176] Selectively expanded cells were stained and evaluated by FACS as described in Example 10. Unstained PBMC ( Fig.19A ) and PBMCs electroporated with electroporation buffer only (mock) (no TCR) ( Fig.19B ) was used as a negative control. The results are shown in Figures 19A-19B In. Figures 19A-19B In the present study, cell counts were determined from a T175 flask on day 14 after one round of expansion with H57. Figures 19A-19B As shown, one round of expansion with H57 resulted in a selective overgrowth of the number of mTCR+ T cells.

[0177] Example 12

[0178] This example demonstrates that further expansion of the number of cells that underwent a first round of expansion with H57 by a second round of expansion with OKT3 in gas permeable flasks resulted in a massive expansion of the number of TCR-transposed T cells.

[0179] PBMCs from donors 1 and 2 were electroporated as described in Example 10. The number of transposed cells was selectively expanded using the H57 antibody as described in Example 11. + After the number of T cells is reached, mTCR is further expanded using standard REP + In standard REP, the number of T cells is selectively amplified. + T cells (5x 10 6 ) and irradiated PBL (5x10 8 ), OKT3 antibody (30 ng / mL) and IL-2 (3000 IU / mL) were co-cultured.

[0180] Selectively expanded cells were stained and evaluated by FACS as described in Example 10. Unstained PBMC ( Fig. 20A ) and PBMCs electroporated with electroporation buffer only (mock) (no TCR) ( Fig. 20B) was used as a negative control. Table 5 shows the cell counts on day 14 after standard REP from one GREX-100 gas permeable flask (Wilson Wolf Corporation, St. Paul, MN). The percentage of mTCR+ cells on day 14 after selective expansion with H57 is shown in Table 6.

[0181] Table 5

[0182]

[0183] Table 6

[0184]

[0185] The results are shown in Figures 20A-20B In. Figures 20A-20B As shown, further expansion of the number of cells that underwent the first round of H57 expansion with a second round of OKT3 expansion in gas permeable flasks resulted in a massive expansion of the number of TCR-transposed T cells.

[0186] Example 13

[0187] This example demonstrates that TCR-transposed T cells selectively expanded with the H57 antibody are specific for the cognate mutated neoantigen.

[0188] 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 mutated HUWE1. 4149-TP53-TCRa2b2 recognizes mutated TP53 (Y220C).

[0189] 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 x 10 4 Peptide-pulsed immature DCs and 10 5 Co-cultures of 10 T cells were incubated overnight at 37° C. DCs cultured alone and DCs pulsed with DMSO were used as controls. IFN-γ secretion was measured by ELISA (enzyme-linked immunosorbent assay).

[0190] The results are shown in Figures 21A-21D In. Figures 21A-21D As shown, TCR-transposed T cells selectively expanded with the H57 antibody recognized the cognate mutated neoantigen.

[0191] Embodiment 14

[0192] This example demonstrates a method to selectively expand the number of T cells expressing mTCR.

[0193] Electroporation of PBMCs with mTCR and selective expansion of mTCR according to one embodiment of the present invention is illustrated. + A schematic diagram of the T cell counting method is shown in Fig. 22 In. Fig. 22 As shown, autologous PBMCs were obtained from patients and cryopreserved. Some PBMCs were depleted of CD4+ cells using LD columns. Cryopreserved PBMCs were thawed in complete medium (CM) and spun at 175g for 10 minutes at approximately 22°C. Cells were washed in Hank's balanced salt solution (HBSS) and counted. The cell count was 6 x 10 7 .

[0194] like Fig. 22 As shown, using AMAXA NUCLEOFECTOR II equipment and kit (Lonza, Basel, Switzerland), the SBTS plasmid encoding mTCR (4149-HUWE1-TCR1 or 4149-TP53-TCRa2b2) and the SBTS plasmid encoding transposase are electroporated into autologous PBMC. Human T cell NUCLEOFECTOR solution (300 μL) contains SBTS plasmid encoding mTCR (45 μg) and SBTS plasmid encoding transposase (15 μg). The human T cell NUCLEOFECTOR solution is added to the cells, and then the mixture of cells and DNA is added to the cuvette (100 μL / cuvette).

[0195] like Fig. 22 As shown, the 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 broad-spectrum nuclease was incubated at 37°C for 1 hour. The cells were harvested, stained and mTCR expression was measured by FACS. A mixed electroporated cell population containing cells expressing mTCR (mTCR+>1%) and cells not expressing mTCR was obtained.

[0196] like Fig. 22 As shown, after culturing at 37°C for more than 18 hours, T cells expressing the mouse TCR constant region (mTCR + In order to selectively amplify mTCR +T cells, electroporated cells (5 x 10 6 ) and irradiated PBL (1x 10 8 ) co-cultivation.

[0197] like Fig. 22 As shown, cells were cultured for 13 days. During this 13 day period, cells were fed with 50 / 50 CM with IL-2 (50 IU / mL) and IL-21 (30 ng / mL) every 2-3 days.

[0198] like Fig. 22 As shown, mTCR+ cells were further enriched by contacting them with microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) conjugated with anti-biotin antibody and biotinylated H57 antibody in LS columns (Miltenyi Biotec).

[0199] After enrichment of mTCR+ cells with H57-conjugated beads, the number of mTCR+ T cells was further expanded using standard REP. + T cells (5x 10 6 ) and irradiated PBL (5x 10 8 ), OKT3 antibody (30 ng / mL) and IL-2 (3000 IU / mL) were co-cultured.

[0200] The timeline followed is as follows:

[0201] Day 0 = electroporation (2x10 7 Total PBMCs (TP53-TCR) or CD4-depleted PBMCs (HUWE1-TCR)

[0202] Day 1 = H57 REP (T175 flask; 5x10 6 cells, 1x10 8 irradiated PBMC, 250ng / mL H57, 50IU / mL IL-2, 30ng / mL IL-21)

[0203] Day 14 = H57 bead enrichment (H57-biotin mAb (GMP), CliniMACS biotin beads, LS column)

[0204] Day 15 = OKT3 REP (GREX-100 flask; 5e6 cells, 5e8 irradiated PBMCs, 30 ng / mL OKT3, 3000 IU / mL IL-2)

[0205] Day 28 = Harvest, phenotype, co-cultivation.

[0206] Embodiment 15

[0207] This example demonstrates the total number of cells and percentage of CD3+mTCR+ cells obtained at four time points in the method described in Example 14.

[0208] 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 the following four time points as described in Example 14: after electroporation, after selective expansion with H57, after enrichment with H57-conjugated beads, and after standard REP with OKT3.

[0209] After electroporation and H57-conjugated bead enrichment, cells were left behind. Counts were not adjusted to reflect theoretical yields, but rather actual yields.

[0210] The results are shown in Fig.23A (total number of cells) and Fig. 23B (Percentage of CD3+mTCR+ cells).

[0211] Example 16

[0212] This example demonstrates the percentage of mTCRβ+ cells measured at day 28 (after OKT3 REP) as described in Example 14.

[0213] 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 middle. Figures 23C-23D PBMCs electroporated with electroporation buffer only (mock) (no TCR) were used as negative control.

[0214] The percentage of CD4+mTCRβ+ or CD8+mTCRβ+ cells was measured by FACS on day 28 (after OKT3 REP). The results are shown in Fig.23E middle.

[0215] Embodiment 17

[0216] This example demonstrates the memory cell phenotype of cells before (day 1) and after (day 28) cell number expansion as described in Example 14.

[0217] The method described in Example 14 was performed. The expression of memory phenotype markers was measured before (day 1) or after (day 28) cell number expansion. The results are shown in Figures 24A-24D middle.

[0218] Embodiment 18

[0219] This example demonstrates the specificity of T cells after expansion of cell numbers as described in Example 14.

[0220] The method described in Example 14 was performed. Immature DCs were pulsed with WT HUWE1 peptide, mutated HUWE1 peptide, WT TP53 peptide, or mutated TP53 peptide at a concentration of 10, 1, or 0.1 μg / mL. Co-cultures of peptide-pulsed immature DCs and transposed T cells were incubated overnight at 37°C. DCs pulsed with DMSO were used as controls. IFN-γ secretion was measured by ELISA. The results are shown in Figures 25A-25B middle.

[0221] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0222] In the context of describing the present invention (particularly in the context of the appended claims), the use of the terms "one" and "a kind of" and "the" and "at least one" and similar references should be interpreted as covering both singular and plural forms, unless otherwise specified herein or clearly contradictory to the context. The term "at least one" is followed by listing one or more (e.g., "at least one of A and B") and should be understood to mean any combination of two or more of one selected from the listed items (A or B) or the listed items (A and B), unless otherwise specified herein or clearly contradictory to the context. Unless otherwise specified, the terms "include", "have", "include" and "contain" should be interpreted as open terms (i.e., meaning "including but not limited to"). Unless otherwise specified herein, the description of the numerical range herein is only intended to be used as a shorthand method for referring to each individual value falling within the range, respectively, and each individual value is incorporated into the specification, just as it is separately set forth herein. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. No language in the specification should be construed as indicating that any unclaimed element is essential to the implementation of the present invention.

[0223] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for carrying out the present invention. After reading the foregoing description, variations of those preferred embodiments will become apparent to those of ordinary skill in the art. The inventors expect that skilled technicians will appropriately adopt such variations, and the inventors wish to implement the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents to the subject matter recorded in the appended claims as permitted by applicable law. In addition, unless otherwise noted herein or clearly contradictory to the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof. Sequence Listing <110> THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES <120> Method for selective expansion of cells expressing TCRs with murine constant regions <130> 740358 <150> US 62 / 568,339 <151> 2017-10-05 <160> 6 <170> PatentIn Version 3.5 <210> 1 <211> 173 <212> PRT <213> House mouse (Mus musculus) <400> 1 Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro 1 5 10 15 Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu 20 25 30 Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys 50 55 60 Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 65 70 75 80 Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe 85 90 95 His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro 100 105 110 Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly 115 120 125 Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu 130 135 140 Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser 145 150 155 160 Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser 165 170 <210> 2 <211> 109 <212> PRT <213> Mus musculus <400> 2 Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro Ser 1 5 10 15 Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu Ala 20 25 30 Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 35 40 45 Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys Glu 50 55 60 Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr 65 70 75 80 Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe His 85 90 95 Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro 100 105 <210> 3 <211> 121 <212> PRT <213> Armenian hamster <400> 3 Glu Val Tyr Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Ser 1 5 10 15 Ser Leu Lys Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Asn Ile Pro Asn Asn Tyr Ala Thr Glu Tyr Ala Asp 50 55 60 Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg Asn Ser 65 70 75 80 Ile Tyr Leu Gln Met Asn Arg Leu Arg Val Asp Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Thr Arg Ala Gly Arg Phe Asp His Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser Ala 115 120 <210> 4 <211> 110 <212> PRT <213> Armenian hamster <400> 4 Tyr Glu Leu Ile Gln Pro Ser Ser Ala Ser Val Thr Val Gly Glu Thr 1 5 10 15 Val Lys Ile Thr Cys Ser Gly Asp Gln Leu Pro Lys Asn Phe Ala Tyr 20 25 30 Trp Phe Gln Gln Lys Ser Asp Lys Asn Ile Leu Leu Leu Ile Tyr Met 35 40 45 Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Thr 50 55 60 Ser Gly Thr Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Pro Glu Asp 65 70 75 80 Glu Ala Ala Tyr Tyr Cys Leu Ser Ser Tyr Gly Asp Asn Asn Asp Leu 85 90 95 Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Arg Gly Pro 100 105 110 <210> 5 <211> 222 <212> PRT <213> Armenian hamster <400> 5 Glu Val Tyr Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Ser 1 5 10 15 Ser Leu Lys Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Asn Ile Pro Asn Asn Tyr Ala Thr Glu Tyr Ala Asp 50 55 60 Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Arg Asn Ser 65 70 75 80 Ile Tyr Leu Gln Met Asn Arg Leu Arg Val Asp Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Thr Arg Ala Gly Arg Phe Asp His Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser Ala Thr Thr Thr Ala Pro Ser Val 115 120 125 Tyr Pro Leu Ala Pro Ala Cys Asp Ser Thr Thr Ser Thr Thr Asp Thr 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ser Val Leu His Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Thr Trp Pro Lys Gln Pro Ile Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg 210 215 220 <210> 6 <211> 212 <212> PRT <213> Armenian hamster <400> 6 Tyr Glu Leu Ile Gln Pro Ser Ser Ala Ser Val Thr Val Gly Glu Thr 1 5 10 15 Val Lys Ile Thr Cys Ser Gly Asp Gln Leu Pro Lys Asn Phe Ala Tyr 20 25 30 Trp Phe Gln Gln Lys Ser Asp Lys Asn Ile Leu Leu Leu Ile Tyr Met 35 40 45 Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Thr 50 55 60 Ser Gly Thr Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Pro Glu Asp 65 70 75 80 Glu Ala Ala Tyr Tyr Cys Leu Ser Ser Tyr Gly Asp Asn Asn Asp Leu 85 90 95 Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Arg Gly Pro Lys Ser 100 105 110 Ser Pro Lys Val Thr Val Phe Pro Pro Ser Pro Glu Glu Leu Arg Thr 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu Val Asn Asp Phe Tyr Pro Gly Ser 130 135 140 Ala Thr Val Thr Trp Lys Ala Asn Gly Ala Thr Ile Asn Asp Gly Val 145 150 155 160 Lys Thr Thr Lys Pro Ser Lys Gln Gly Gln Asn Tyr Met Thr Ser Ser 165 170 175 Tyr Leu Ser Leu Thr Ala Asp Gln Trp Lys Ser His Asn Arg Val Ser 180 185 190 Cys Gln Val Thr His Glu Gly Glu Thr Val Glu Lys Ser Leu Ser Pro 195 200 205 Ala Glu Cys Leu 210

Claims

1. A method for selectively expanding a plurality of T cells, the method comprising: modifying a human T cell to express a TCR, wherein the TCR comprises a murine constant region; Producing a cell population comprising a plurality of said human T cells expressing said TCR and a plurality of human T cells not expressing said TCR; and culturing the cell population in the presence of (i) irradiated feeder cells, (ii) (a) IL-2 or (b) IL-2 and IL-21, and (iii) a first antibody or an antigen-binding portion thereof, The first antibody or its antigen binding portion can specifically bind to the mouse constant region of the TCR so as to selectively expand the number of human T cells expressing the TCR over the number of human T cells not expressing the TCR.

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 antigenic specificity for a viral antigen.

4. The method of any one of claims 1-3, further comprising culturing the cell population in the presence of (i) one or both of irradiated allogeneic feeder cells and irradiated autologous feeder cells, (ii) IL-2, and (iii) a second antibody that specifically binds to a human CD3 complex, or an antigen-binding portion thereof.

5. The method according to any one of claims 1-3, wherein the first antibody can specifically bind to the mouse constant region of the β chain of the TCR.

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

4.

7. The method of any one of claims 1-3, wherein modifying human T cells to express the TCR comprises modifying the human T cells to express the TCR using transfection, transformation, transduction, electroporation, transposon, meganuclease, zinc finger nuclease, transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system.

8. The method of any one of claims 1-3, comprising increasing the number of T cells expressing a TCR comprising a murine constant region by 10-fold to 1,000-fold.

9. The method of any one of claims 1-3, wherein the TCR comprises a murine variable region.

10. The method of any one of claims 1-3, wherein the TCR comprises a human variable region.

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