Immune cells for adoptive cell therapy

Abstract

By engineering BCL6 and cell survival-promoting genes to express, methods are provided for generating infinite immune cells with long life spans and high proliferation rates. Further provided herein are methods for generating the infinite immune cells and methods for using the infinite immune cells to treat diseases such as cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 889,662, filed August 21, 2019, which is incorporated herein by reference in its entirety. [Technical Field]

[0002] The present disclosure relates generally to at least the fields of molecular biology, cell biology, immunology, and medicine. More particularly, the present disclosure relates to methods for generating and using unlimited immune cells. [Background technology]

[0003] NK cells and T cells are two types of cytotoxic lymphocytes commonly used in adoptive cell therapy research. NK cell- and T cell-derived CAR-NK cells, CAR T cells, TCR-transduced T cells, and T cells with endogenous T cell receptors specific for microbial or tumor antigens are highly promising approaches for the treatment of both hematological and solid tumors. Three CAR-T cell products targeting CD19 have recently been approved by the FDA for use in B cell malignancies, and many more are in development. Currently, the production of both TCR-T cell and CAR-T cell therapy products is a multistep process that requires the isolation of T cells from healthy donors or patients, the introduction of TCRs or CARs into those T cells using viral or nonviral vectors, and the in vitro expansion of the genetically modified T cells before infusion into the patient. The generation of T cells specific for microbial and tumor antigens is similarly a multi-step process that requires the collection of T cells from a healthy donor or patient, followed by in vitro isolation and / or stimulation with microbial or tumor antigen peptides or proteins, and in vitro expansion of the T cells before infusion into the patient.

[0004] Therefore, the production of individual patient products is costly, cumbersome, and time-consuming. Moreover, the T cells thus produced can only be expanded in vitro for a few weeks before senescence, limiting the number of microbial antigen-specific and tumor antigen-specific T cells, TCR-T cells, or CAR-T cells that can be produced from each patient or healthy donor.

[0005] A recent report suggested that factors that promote the survival of CAR-T cells through genetic manipulation are positively associated with better therapeutic outcomes (Hurton et al., 2016). Therefore, strategies that extend the lifespan of normal and / or genetically modified T cells, as well as strategies that preserve their proliferation, cytokine production, and cytotoxic functions, may enhance the efficacy of these cells while significantly shortening the time and cost of adoptive T cell therapy approaches. The cytotoxic T cell line TALL-104 (U.S. Patent No. 5,272,082) and the NK cell line NK-92 (U.S. Patent Application Publication No. 20020068044) are capable of indefinite proliferation and possess cytotoxic activity, but were established from T cell leukemia and NK cell leukemia, respectively. Therefore, these cell lines contain mutations and other genetic alterations that make them unsafe for use in human therapy. Therefore, there is an unmet need for strategies that achieve these goals of extending the lifespan of normal T cells. Summary of the Invention

[0006] In one embodiment, the present disclosure provides a composition comprising immune cells (including at least T cells or NK cells) engineered to have a longer lifespan compared to non-engineered immune cells. Such cells may be referred to herein as immortal cells. In a specific embodiment, the methods and compositions relate to immune cells having expression (including heterologous expression) of B-cell lymphoma 6 (BCL6) and a pro-survival gene or an anti-apoptotic gene or a cell survival-promoting gene. As used herein, a pro-survival gene refers to a nucleic acid polymer capable of exerting an anti-apoptotic function or promoting survival by any mechanism. The nucleic acid polymer capable of exerting an anti-apoptotic function may be one or more Bcl2 family genes (e.g., BCL-xL (also known as the BCL2L1 gene), BCL-2, MCL1, BCL2L2 (Bcl-w), BCL2A1 (Bfl-1), BCL2L10 (BCL-B), etc.). The nucleic acid polymer capable of exerting anti-apoptotic function may be one or more inhibitor of apoptosis (IAP) family genes (e.g., XIAP, BIRC2 (C-IAP1), BIRC3 (C-IAP2), NAIP, BIRC5 (survivin), etc.). The nucleic acid capable of exerting anti-apoptotic function may be capable of inhibiting or knocking out the expression of one or more caspases that play a role in apoptosis (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14). The nucleic acid polymer for knockdown or knockout may be an shRNA expression cassette, or these caspase genes may also be knocked out by gene editing methods (CRISPR, TALEN, zinc finger method, etc.). Nucleic acid polymers capable of exerting anti-apoptotic function may be able to inhibit or knock out the expression of one or more pro-apoptotic genes (e.g., BCL2L11 (BIM), BBC3 (PUMA), PMAIP1 (NOXA), BIK, BMF, BAD, HRK, BID, BAX, BAK1, BOK, etc.).Nucleic acid polymers capable of exerting anti-apoptotic functions may have anti-apoptotic effects (e.g., IGF1, HSPA4 (Hsp70), HSPB1 (Hsp27), CLAR (cFLIP), BNIP3, FADD, AKT and NF-κB, RAF1, MAP2K1 (MEK1), RPS6KA1 (p90Rsk), JUN, c-Jun, BNIP2, BAG1, HSPA9, HSP90B1, miRNA21, miR-106b-25, miR-206, miR-221 / 222, miR-17-92, miR-133, miR-143, miR-145, miR-155, miR-330, etc.).

[0007] In certain embodiments, the cells encompassed herein can constitutively produce large amounts of IL-4 in the absence of external stimuli (e.g., greater than 1000 pg / mL in in vitro cultures when incubated at a cell concentration of 10,000 cells / mL), and such cells can be used in clinical applications (e.g., to treat various inflammatory disorders, including autoimmune diseases, graft-versus-host disease, certain types of infections associated with cytokine release syndrome, toxicities associated with CAR T cell therapy and other adoptive T cell therapies, inflammatory bowel disorders, immune-related adverse events associated with various immunotherapies, hemophagocytic lymphohistiocytosis, periodic fever syndromes, etc.) because IL-4 can suppress inflammation induced by T cells, macrophages, and other immune cells.

[0008] In some embodiments, the cell survival-promoting gene is an anti-apoptotic B-cell lymphoma 2 (BCL-2) family gene.In certain embodiments, the anti-apoptotic BCL-2 family gene is BCL2L1 (Bcl-xL), BCL-2, MCL1, BCL2L2 (Bcl-w), BCL2A1 (Bfl-1), BCL2L10 (BCL-B) or a combination thereof.In certain embodiments, the anti-apoptotic BCL-2 family gene is Bcl-xL.

[0009] In a further embodiment, the T cells or NK cells are further engineered to express IL-2 and / or IL-15.

[0010] In certain embodiments, the T cells or NK cells are derived from a healthy donor (e.g., a donor who has not been diagnosed with cancer). In other embodiments, the T cells or NK cells are derived from a patient. In certain embodiments, the donor is human.

[0011] In specific embodiments, the T cells include CD4+ T cells, CD8+ T cells, iNKT cells, NKT cells, γδ T cells, regulatory T cells, innate lymphoid cells, or a combination thereof. In some embodiments, the T cells include CD8 and / or γδ T cells. The T cells are naive T cells, effector T cells, memory T cells, stem cell memory T cells, terminally differentiated T cells, or a combination thereof. In certain embodiments, the T cells are TCRαβ cells or TCRγδ T cells. In some embodiments, the composition does not include or is essentially free of follicular helper (Tfh) T cells. In some embodiments, the composition of immune cells is T cells that are Th1 / Tc1, Th2 / Tc2, Th9 / Tc9, Th17 / Tc17, Tfh, Th22, Tc22, or a combination thereof. In certain embodiments, the T cells express IFNγ, granzyme B, perforin, or a combination thereof.

[0012] In certain embodiments, the T cells or NK cells are virus-specific or tumor antigen-specific. In some embodiments, the T cells or NK cells are further engineered to express one or more CARs and / or one or more TCRs. In some embodiments, the CARs or TCRs are CD4, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD79a, CD79b, SLAM-F7, CD123, CD70, CD72, CD33, CD38, CD80, CD86, CD138, CLL-1, FLT3, ROR-1, TACI, TRBC1, MUC1, PD-L1, CD117, F In certain embodiments, the CAR comprises a CD19 antigen binding region.

[0013] In certain embodiments, the composition comprises at least 50 million, 100 million, 200 million, 500 million, 750 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, or 10 billion immune cells (including T cells, innate lymphoid cells, NK cells, or a mixture thereof).

[0014] In additional embodiments, the immune cells comprise at least one safety switch. In some embodiments, the safety switch is a truncated EGFR (e.g., an EGFR lacking domains 1 and 2). In some embodiments, the immune cells (T cells, innate lymphoid cells and / or NK cells) express IL-2, IL-15, other growth or differentiation factors, or a combination thereof.

[0015] In some embodiments, the cells maintain a proliferation rate for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or any range therebetween. In certain embodiments, the immune cells have enhanced anti-tumor cytotoxicity, in vivo proliferation, in vivo persistence, and / or improved function.

[0016] In another embodiment, a method for producing T cells, innate lymphoid cells, or NK cells of this embodiment is provided, the method comprising introducing into the cells a vector encoding BCL6 and a cell survival-promoting gene. In some embodiments, the cell survival-promoting gene is an anti-apoptotic B-cell lymphoma 2 (BCL-2) family gene. In some embodiments, the anti-apoptotic BCL-2 family gene is BCL2L1 (Bcl-xL), BCL-2, MCL1, BCL2L2 (Bcl-w), BCL2A1 (Bfl-1), or BCL2L10 (BCL-B). In a specific embodiment, the anti-apoptotic BCL-2 family gene is Bcl-xL. In a specific embodiment, the vector links BCL6 and Bcl-xL with a 2A sequence. In a specific embodiment, the 2A sequence is a T2A sequence.

[0017] In some embodiments, the vector is a lentiviral vector. In certain embodiments, the introducing step comprises transducing the cells with the lentiviral vector in the presence of IL-2 and / or other growth factors. In certain embodiments, IL-2 is present at a concentration of 10 IU / mL to 1000 IU / mL, e.g., 10 to 50 IU / mL, 50 to 75 IU / mL, 75 to 100 IU / mL, 100 to 250 IU / mL, 250 to 500 IU / mL, 500 to 750 IU / mL, or 750 to 1000 IU / mL. In certain embodiments, IL-2 is present at a concentration of 100, 200, 300, 400, or 500 IU / mL.

[0018] In additional embodiments, the method further comprises activating the T cells with CD3 and CD28. In some embodiments, the method further comprises culturing the cells in the presence of IL-2 and / or IL-15. In certain embodiments, IL-2 and / or IL-15 are present at a concentration of 10 ng / mL, 25 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL. In some embodiments, the cells are cultured for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months (or any range therebetween) without essentially a decrease in proliferation rate.

[0019] In further embodiments, the method further comprises sorting for a T cell subset. In particular embodiments, the T cell subset comprises CD4+ T cells, CD8+ T cells and / or γδ T cells.

[0020] Embodiments include compositions comprising the cell populations of the embodiments (e.g., immune cells engineered to express B-cell lymphoma 6 (BCL6) and cell survival-promoting genes) for treating immune-related disorders, infectious diseases and / or cancer.

[0021] Embodiments relate to methods of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an immune cell of the embodiments (e.g., an immune cell engineered to express B-cell lymphoma 6 (BCL6) and a cell survival-promoting gene).

[0022] In some embodiments, the disease or disorder is an infectious disease, cancer, and / or immune-related disorder. In certain embodiments, the immune-related disorder is an autoimmune disorder, graft-versus-host disease, allograft rejection, or other inflammatory condition. In some embodiments, the immune cells are allogeneic. In certain embodiments, the immune-related disorder is cancer. For example, the cancer is a solid cancer or a hematological malignancy.

[0023] In additional embodiments, the method further comprises administering at least a second therapeutic agent. In some embodiments, the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy, drug therapy, hormone therapy, biological therapy, or a combination thereof.

[0024] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0025] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0026] [Figure 1A] Map of the lentiviral vector containing the BCL6-T2A-BCL-xL gene driven by the human PGK promoter. [Figure 1B]Graph showing the proliferation rates of infinite T cell lines. The top left panel shows the growth curves of In1-L4a (infinite CD3 T cells) and Ie1-L4aJ3 (infinite CD8 CAR-T) in the presence of 400 IU / mL of IL-2 at 2 months. The top right panel shows the growth curve of Infinite CD8 CAR T cells (Ie1-L4aJ3) in the presence of 100 ng / mL of IL-15, IL-7, and IL-21, or in the absence of cytokines. The data show that Infinite T cells grow in the presence of IL-15, but not in the presence of IL-7, IL-21, or in the absence of cytokines. The bottom left and right panels show that infinite T cells, including CD4 infinite αβ T cells, CD8 infinite αβ T cells (Ie1-L4a), CD8 infinite αβ CAR-T cells (Ie1-L4aJ3), infinite γδ T cells (Igd1-L4a), and infinite γδ CAR-T cells (Igd1-L4aJ3), continue to expand in vitro in the presence of IL-2 at 5 months. [Figure 1C] Graph showing the phenotype of the infinite T cell line In1-L4a as measured by expression of CD3, CD4, CD8, CD16, CD56, TCRαβ, and TCRγδ. [Figure 1D] Graph showing the phenotype of γδ T cells sorted using anti-TCRγδ antibodies, showing the expression of TCRγδ, TCRαβ, and CD16 on these cells. [Figure 1E] Graph showing the major subsets of γδ T cells sorted using anti-TCRγ9 and anti-TCRδ2 antibodies. The majority of unbound γδ T cells are TCRγ9δ2 positive. [Figure 1F] Graph showing the phenotype of infinite T cells at 4 months, the majority of which are effector and central memory T cells that primarily express IFNγ, granzyme B, and perforin. [Figure 1G] Graph showing the expression of various co-inhibitory receptors on Infinite CAR-T cells.

[0027] [Figure 2A]Map of lentiviral vector pJ3 containing an anti-CD19 CAR and a truncated human EGFR expression cassette. [Figure 2B] Graph showing the CAR-positive percentage of Ie1-L4aJ3 (infinite CD8 CART) and In1-L4aJ3 (infinite CD3 CART) transduced with the lentiviral vector pJ3. The CAR-positive percentage was measured by flow cytometry using FITC-labeled human CD19 protein or anti-EGFR antibody 10 days after transduction. [Figure 2C] Graph showing the CAR-positive cell percentage of In1-L4aJ3 (infinite CD3 CART). tEGFR was stained with AF647-labeled cetuximab, and anti-CD19 CAR was stained with FITC-labeled recombinant human CD19 protein. [Figure 2D] Graph showing the percentage of CAR-positive cells for In1-L4aJ3 (infinite CD3 CART) before and after sorting. tEGFR was stained with AF647-cetuximab, and anti-CD19 CAR was stained with FITC-labeled recombinant human CD19 protein.

[0028] [Figure 3] Graph showing the in vitro cytotoxicity of Ie1-L4aJ3 (infinite CD8 CART) against Raji and Nalm6 cells at effector:target (E:T) ratios of 0.2:1 and 1:1 in 12-well plates. Ie1-L4aJ3 (infinite CD8 CART) cells or control Ie1-L4a (infinite CD8 T cells without a CAR) cells were cocultured with Raji or Nalm6 cells for 5 days. The percentage of tumor cells in the coculture on days 0, 1, 3, and 5 is shown.

[0029] [Figure 4]Graph showing in vitro cytotoxicity of infinite T cells after 4 months of expansion. Ie1-L4a (infinite CD8 T cells), Ie1-L4aJ3 (infinite CD8 CAR-T cells), Igd1-L4a (infinite gamma / delta T cells), or Igd1-L4aJ3 (infinite γδ CAR-T cells, CAR-T percentage >90%) cells were cocultured with Daudi or Nalm6 cells at an effector:target (E:T) ratio of 3:1 in 12-well plates in the presence of IL-15 for 7 days. The percentage of tumor cells in the coculture on days 0, 1, 2, 4, and 7 is shown. These results suggest that 1) CD8-infinite CAR-T and γδ-infinite CAR-T cells maintained their specific cytotoxicity even after long-term in vitro culture and expansion, and 2) γδ-infinite T cells lacking a CAR but bearing an endogenous γ9δ2 TCR or other TCRs can induce lysis of certain types of tumor cells, likely mediated by the γδ TCR. For example, Daudi cells can be killed by γδ-infinite T cells lacking a CAR, whereas Nalm-6 cells can only be killed by γδ-infinite T cells transduced with a CAR. In addition to some lymphoma cells, some myeloma and other cancer cell lines are also known to be killed by γδ T cells.

[0030] [Figure 5A] Growth kinetics of infinite T cells (CD4+CD8 or CD8) with or without anti-CD19 CAR in the presence of IL-2. [Figure 5B] The infinite T cells have a mixture of both CD4 and CD8 T cells (left panel) and can be sorted to high purity as shown for CD8 infinite T cells (right panel). [Figure 5C] Infinite T cells were then incubated without IL-2 (shown) or IL-15 (not shown) in culture for 6 months. Cell numbers rapidly decreased within 6 days, suggesting that there was no evidence of autonomous proliferation or malignant transformation of infinite T cells even after long-term in vitro culture.

[0031] [Figure 6A] Telomerase activity in unlimited T cells or peripheral blood mononuclear cells (PBMCs) was measured using the TRAPeze Telomerase Activity Detection Kit according to the manufacturer's instructions. [Figure 6B] Genes associated with telomerase activity, as measured by RNAseq analysis, in infinite T cells or corresponding PBMC samples, shown as a heatmap. These results suggest that infinite T cells have very high telomerase activity.

[0032] [Figure 7A] Infinite T cells with or without anti-CD19 CARs, or CAR T cells generated by conventional methods from peripheral blood T cells, were labeled with CellTrace FarRed and co-cultured with Daudi tumor cells labeled with CellTrace Violet at a 1:1 effector:target ratio. The percentage of viable tumor cells (lower right gate) was measured after 3, 5, and 7 days. Absolute numbers of viable tumor cells were also calculated by flow cytometry using CountBright Absolute counting beads (ThermoFisher Scientific), and these results were consistent with the percentages of viable tumor cells shown. [Figure 7B] Infinite T cells with or without anti-CD19 CAR were cocultured 1:1 with NALM-6 B cell leukemia cells. Degranulation was measured after 6 hours by CD107a staining. These results suggest that CAR-expressing Infinite T cells are highly cytotoxic and degranulate in response to B cell tumors. [Figure 7C] The phenotype of anti-CD19 infinite CAR T cells was measured for the markers shown by flow cytometry.The expression of anti-CD19 CAR was measured by staining with fluorescently labeled recombinant human CD19-Fc protein.These results show that infinite T cells do not express high levels of traditional exhaustion markers (e.g., CTLA-4, PD-1, TIM-3, CD160 or 2B4 (CD244)). [Figure 7D] The phenotype of anti-CD19 infinite CAR T cells was measured for the markers shown by flow cytometry.The expression of anti-CD19 CAR was measured by staining with fluorescently labeled recombinant human CD19-Fc protein.These results show that infinite T cells do not express high levels of traditional exhaustion markers (e.g., CTLA-4, PD-1, TIM-3, CD160 or 2B4 (CD244)).

[0033] [Figure 8A] Genes or gene signatures associated with T cell subsets shown as heatmaps in unbound T cells or corresponding PBMC samples, as determined by RNAseq analysis. [Figure 8B] Genes or gene signatures associated with exhaustion markers in infinite T cells or corresponding PBMC samples, as measured by RNAseq analysis, shown as heatmaps. [Figure 8C] Genes or gene signatures related to chemokine receptors shown as heatmaps in infinite T cells or corresponding PBMC samples, as determined by RNAseq analysis. [Figure 8D] Genes or gene signatures associated with senescence markers shown as heatmaps in infinite T cells or corresponding PBMC samples, as measured by RNAseq analysis.

[0034] [Figure 9A] Genes associated with chemokine expression, shown as a heatmap, in infinite T cells or corresponding PBMC samples, as determined by RNAseq analysis. [Figure 9B] Genes involved in cytokine expression, shown as heatmaps, in infinite T cells or corresponding PBMC samples, as measured by RNAseq analysis. [Figure 9C]Genes related to cytokine receptors, shown as heatmaps, in infinite T cells or corresponding PBMC samples, as determined by RNAseq analysis.

[0035] [Figure 10A] Infinite T cells or CAR-transduced T cells were thawed, and expression of anti-CD19 CAR was measured by anti-EGFR antibody staining. [Figure 10B] Growth kinetics of anti-CD19-infinite CAR T cells after thawing and culturing with IL-2 in vitro. The number of cells in culture at various days is shown. [Figure 10C] The cytotoxic activity of the thawed cells in A was measured 4 days after 1:1 coculture of infinite T cells with NALM-6 tumor cells as described under Figure 7A. The gate indicates the percentage of viable tumor cells.

[0036] [Figure 11] The phenotype of limitless γδ T cells (bottom) was measured for indicated markers by flow cytometry and compared with corresponding γδ T cells (top) derived from healthy donor PBMCs. These results demonstrate that limitless γδ T cells do not express high levels of traditional exhaustion markers.

[0037] [Figure 12] Luciferase-labeled infinite T cells were injected intraperitoneally (ip) with or without IL-15 on days 1 and 3. T cell numbers were imaged by bioluminescence imaging (BLI). These results demonstrate that IL-15 promotes the growth and expansion of infinite T cells in vivo.

[0038] [Figure 13]Luciferase-labeled NALM-6 cells were injected into NSG mice with + / -IL-15 along with infinite T cells with or without anti-CD19 CAR. Anti-tumor efficacy was measured by BLI (left) and survival (right). These results demonstrate that anti-CD19 infinite CAR T cells have anti-tumor efficacy in vivo.

[0039] [Figure 14] Antigen-specific Infinite T cells. Infinite T cells from HLA-A2+ donors were tested for specificity against infectious disease- and tumor-associated antigens using HLA-A2 tetramers bearing known CD8 T cell epitopes. The data indicate that among the Infinite T cells, there are antigen-specific T cells that recognize microbial and tumor-associated antigens via their endogenous TCRs.

[0040] [Figure 15] Generation of EBV-specific infinite T cells. Healthy donor peripheral blood mononuclear cells from HLA-A2+ donors were stimulated with a pool of EBV peptides that bind to HLA-A2 on day 0. 24 hours later, CD137-positive T cells were sorted by flow cytometry and used to generate infinite T cells as previously described by transducing with BCL6 and Bcl-xL. After 7 weeks of culture, tetramer-positive cells were enriched using magnetic beads. The enriched cells were then cultured for another 6 weeks and stained for CD8 and the BMLF1-HLA-A2 tetramer specific for the HLA-A2-binding peptide (GLCTLVAML) derived from the EBV-BMLF1 protein. These results suggest that enriched populations of microbial- or tumor-specific infinite CD4 or CD8 T cells can be generated using the described method.

[0041] [Figure 16]We generated infinite αβ or γδ T cells using the BCL6 and BCL2L1 genes under the control of a Tet-off safety switch. The growth rate of infinite T cells with IL-2 in the presence (right) or absence (left) of 1 μg / mL doxycycline (Dox) is shown. These results suggest that infinite T cells maintained their growth rate in the absence of doxycycline, but stopped proliferating and gradually underwent cell death in the presence of doxycycline. Similar Tet-off safety switches can also be used to control the IL-2 or IL-15 cytokine genes engineered into infinite T cells.

[0042] [Figure 17] Infinite T cells with a tet-off safety switch were cultured with IL-2 in the presence or absence of increasing concentrations of doxycycline (Dox), and the cells in culture were imaged by light microscopy. After two weeks, the cells were stained and CD25 expression was assessed by flow cytometry. Light microscopy imaging revealed that the infinite T cells gradually decreased in size, along with a decrease in proliferative clusters, with increasing concentrations of doxycycline. Furthermore, CD25 expression was significantly reduced in the presence of doxycycline.

[0043] [Figure 18] Infinite T cells with a tet-off safety switch were cultured with IL-2 in the presence or absence of 1 μg / mL doxycycline (Dox). After 2 weeks, cells were stained and evaluated by flow cytometry for the indicated surface markers. PD-1 expression was significantly increased in the presence of doxycycline.

[0044] [Figure 19]Cytokine production by Infinite T cells. Infinite T cells (CD8+) with or without anti-CD19 CAR expression were cocultured with NALM-6 tumor cells at an effector:target ratio of 5:1. After three days, cytokine levels in the supernatant were measured. Data are representative of results from Infinite T cells derived from three different healthy donors. These results show that Infinite T cells with anti-CD19 CARs produced significant amounts of IL-2, GM-CSF, IFNγ, IL-5, and IL-17 in response to NALM-6 tumor cells, whereas Infinite T cells without anti-CD19 CARs did not. Production of TNFα, IL-4, IL-6, IL-10, or IL-13 by anti-CD19 Infinite CAR T cells in response to tumor cells was minimal or not significantly different from Infinite T cells without CAR expression. However, we observed that infinite T cells with or without CAR expression produced large amounts of IL-4, exceeding 10,000 pg / mL, in the presence or absence of tumor cells (Figure 19 and data not shown).

[0045] [Figure 20] Lysis of Infinite CAR T cells by cetuximab via antibody-dependent cell-mediated cytotoxicity (ADCC). Infinite T cells expressing anti-CD19 CAR and tEGFR were labeled with CFSE and co-cultured in duplicate with or without NK cells derived from healthy donors at the indicated effector:target ratios in the presence of 5 μg / mL cetuximab or rituximab. After 5 hours, the absolute number of Infinite T cells in each well was determined by flow cytometry using counting beads, and the percent reduction in Infinite T cell numbers compared to T cells alone was calculated and plotted. The percent reduction in T cells with cetuximab or rituximab in the absence of NK cells was <5%.

[0046] [Figure 21A]Generation of infinite T cells using BCL6 and BCL2L1 genes or BCL6 and BIRC5 (survivin) genes, a Tet-off safety switch, and IL-15. Design of lentiviral constructs containing BCL6 and BCL2L1 genes or BCL6 and BIRC5 genes, a Tet-off safety switch, and IL-15 gene. [Figure 21B] Generation of infinite T cells using BCL6 and BCL2L1 genes or BCL6 and BIRC5 (survivin) genes, a Tet-off safety switch, and IL-15. Human T cells were lentivirally transduced with the constructs shown in panel A and cultured in the presence of IL-2. After 12 weeks, the growth rates of T cells generated by the two approaches were measured in in vitro culture under similar conditions. [Figure 21C] Generation of infinite T cells using BCL6 and BCL2L1 genes or BCL6 and BIRC5 (survivin) genes, a Tet-off safety switch, and IL-15. Lentiviral constructs containing the BCL6 and BCL2L1 genes shown in panel A were used to generate infinite T cells from two donors and cultured with IL-2 in the presence or absence of 1 μg / mL doxycycline. The cells grew at an exponential rate in the absence of doxycycline, but stopped proliferation and gradually underwent cell death in the presence of doxycycline.

[0047] [Figure 22] An example of a construct containing BCL6 together with Bcl-xL (L5x(MSCV-BCL6-P2A-BCL-xL-T2A-rtTA)). This construct contains at least a P2A element between wild-type BCL-6 and BCL-xL, and a T2A element between BCL-xL and rtTA (Tet on transactivator).

[0048] [Figure 23]Illustrative examples of specific embodiments of constructs including those for at least BCL6 expression. Some embodiments include shRNA of any kind, including, by way of example, those against caspase 9 or BAK. Detailed Description of the Invention

[0049] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Ectopic expression of the human telomerase reverse transcriptase (hTERT) gene has previously been reported to immortalize normal T cells (Hooijberg et al., 2000). However, overexpression of hTERT alone has been observed to be insufficient for immortalizing T lymphocytes. Indeed, T cells generated by this approach cease proliferation after a period of time (Migliaccio et al., 2000). This study investigated whether expression of BCL6 in normal NK or T cells could arrest their differentiation and whether expression of pro-survival genes, such as anti-apoptotic BCL-2 family genes like BCL2L1, which encodes the Bcl-xL protein, could significantly extend their lifespan, potentially immortalizing them while maintaining their basic functions.

[0050] Embodiments of the present disclosure relate to compositions, production, and uses of cells that have a significantly extended lifespan compared to cells lacking the modifications encompassed herein. In specific embodiments, the cells encode heterologous BCL6 and one or more pro-survival genes (or anti-apoptotic or cell survival-promoting genes), including any gene whose gene product has anti-apoptotic function. By way of example, the pro-survival gene may be any BCL-2 family gene, including, by way of example only, BCL-xL, BCL-2, MCL-1, or survivin. Additionally or alternatively, the cells have inhibited or knocked out expression of one or more caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, or a combination thereof). In such an example, the DNA fragment for knocking down or knocking out one or more caspase genes can be an shRNA expression cassette.These caspase genes can also be knocked out by gene editing methods (CRISPR, TALEN, zinc finger method, etc.).Therefore, in a specific embodiment, the immune cells contain BCL6 overexpression or heterologous BCL6 plus caspase knockout to create infinite immune cells.These cells can have one or more pro-survival genes (or anti-apoptotic genes or cell survival-promoting genes), and in specific cases, can also have one or more caspase gene knockdown or knockout.

[0051] In certain embodiments, the present disclosure provides methods for generating infinite numbers of immune cells that have a greatly extended lifespan and can be rapidly expanded to large numbers, such as for adoptive immunotherapy. The methods provide, in at least some cases, infinite immune cells that have the capacity to expand indefinitely with a single transduction. The methods are very inexpensive and can generate infinite numbers of immune cells in a short period of time (e.g., one month or more).

[0052] The platform and system encompassed herein can be used to generate limitless immune cells, such as limitless T cells, including both TCRαβ and TCRγδ T cells. This approach provides a limitless source of human T cells that can be used directly or further genetically engineered to generate desired cells, including universal chimeric antigen receptor (CAR) T cells or T cell receptor (TCR)-transduced T cells. In specific embodiments, the cells are used to treat or prevent cancer and other diseases, including infectious and inflammatory disorders. By way of example, the system can be used to treat cancer, infectious diseases, and / or inflammatory diseases. Specific examples include B-cell lymphoma, CMV infection, EBV infection, autoimmune disorders, graft-versus-host disease, or a combination thereof.

[0053] As an example, studies included herein demonstrated that transduction of infinite T cells with an anti-CD19 CAR generated "anti-CD19 infinite CAR T cells" (CD19 inCART) with redirected specificity for human B cell tumors. The CD19 infinite CAR T cells could serve as a source for generating an infinite number of antigen receptor-modified T cells (e.g., CAR T cells) after just one transduction and showed significant cytotoxicity against human B cell lymphoma cell lines. The present disclosure provides a universal immune cell therapy platform and system capable of generating an infinite number of immune cells, dramatically reducing the cost of adoptive immune cell therapy by streamlining the manufacturing process, and dramatically shortening the time to generate the cells. In certain embodiments, expression of BCL6 and one or more pro-survival genes (or anti-apoptotic or pro-cell survival genes) enables the generation of infinite cells, which then become universal cells for further manipulation toward adoptive cell therapy (e.g., by incorporating an engineered antigen receptor of interest (e.g., to adapt to a specific cancer)). The universal cells may also already contain one or more safety switches (e.g., an induction system and an elimination gene, e.g., a truncated EGFR (e.g., lacking domain 1 and / or domain 2) and / or one or more suicide genes and / or one or more cytokines), or any of these may be added later in a process to adapt the cells to have desired properties.

[0054] I. Definition As used herein, "essentially free" of a specific component means that the specific component is not intentionally formulated into the composition and / or is not present even as a contaminant or in trace amounts.Therefore, the total amount of the specific component due to any unintentional contamination of a composition is less than 0.05%, preferably less than 0.01%.Most preferred is a composition in which the amount of the specific component cannot be detected by standard analytical methods.

[0055] As used herein, "a" or "an" can mean one or more. When used in the claims, the words "a" or "an," when used in conjunction with the word "comprising," can mean one or more than one. Some embodiments of the present disclosure can consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments can be combined.

[0056] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, although the present disclosure supports the definition of referring to alternatives only and "and / or." For example, "x, y and / or z" can refer to "x" only, "y" only, "z" only, "x, y and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is expressly intended that x, y, or z can be explicitly excluded from an embodiment. As used herein, "another" can mean at least a second or more. The terms "about," "substantially," and "approximately" generally mean the stated value plus or minus 5%.

[0057] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" are understood to imply the inclusion of the recited step or element or steps or elements, but not the exclusion of any other step or element or steps or elements. "Consisting of" means "including and limited to," regardless of what precedes the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are essential or required, and that other elements may not be present. "Consisting essentially of" means including any elements listed before the phrase, and limited to other elements that do not interfere with or contribute to the activity or action set forth in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are essential or required, but that other elements are not optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0058] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0059] "Immune disorder," "immune-related disorder," or "immune-mediated disorder" refers to a disorder in which the immune response plays a significant role in the development or progression of the disease. Immune-mediated disorders include autoimmune disorders, allograft rejection, graft-versus-host disease, and inflammatory and allergic conditions.

[0060] An "immune response" is a response of a cell of the immune system, such as a B cell or T cell or innate immune cell, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response").

[0061] "Autoimmune disease" refers to a disease in which the immune system mounts an immune response (e.g., a B cell response or a T cell response) against antigens that are part of the normal host (i.e., self-antigens), resulting in tissue damage. Self-antigens can be derived from host cells or from commensal organisms (e.g., microorganisms that normally colonize mucosal surfaces, known as commensals).

[0062] "Treating" a disease or condition or its treatment refers to carrying out a protocol that may include administering one or more drugs to a patient with the aim of alleviating the signs or symptoms of the disease. Desirable effects of treatment include slowing the rate of disease progression, reversing or alleviating the disease state, and alleviating or improving prognosis. Alleviation can occur before or after the signs or symptoms of the disease or condition appear. Thus, "treating" or "treatment" can include "preventing" or "prevention" of a disease or undesirable condition. Furthermore, "treating" or "treatment" does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0063] As used throughout this application, the term "therapeutic effect" or "therapeutically effective" refers to anything that enhances or improves the well-being of a subject with respect to medical treatment of that condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer can include, for example, reducing tumor size, reducing the invasiveness of a tumor, reducing the rate of cancer growth, or preventing metastasis. Treating cancer can also refer to extending the survival time of a subject with cancer.

[0064] "Subject," "patient," and "individual" may be interchangeable and may refer to humans or non-humans, e.g., primates, mammals, and vertebrates. In certain embodiments, the subject is a human. A subject may be any organism or animal subject that is the subject of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject may be a patient and may have or be suspected of having, for example, a disease (which may be referred to as a condition), such as one or more infectious diseases, one or more genetic disorders, one or more cancers, or any combination thereof. A "subject" or "individual," as used herein, may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. Individuals may receive one or more medical compositions via the Internet.Individuals may include humans or non-human animals of any age, so individuals include adults and young people (i.e., children) and infants, and also include individuals in utero.Subjects may or may not need medical treatment;Individuals may be voluntarily or involuntarily part of an experiment, whether clinical or supporting basic scientific research.

[0065] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered appropriately to animals, such as humans. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients will be known to those of skill in the art in light of the present disclosure. Furthermore, it is understood that for administration to animals (e.g., humans), preparations should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0066] As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, flow and nutritional supplements, such similar materials, and combinations thereof, as known to those skilled in the art. The pH of the pharmaceutical composition and the exact concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0067] II. Infinite immune cells Certain embodiments of the present disclosure relate to immune cells engineered to express one or more genes, which directly or indirectly result in an increased lifespan of the cells compared to cells lacking expression of the one or more genes. In certain embodiments, the cells are engineered to express one or more genes, including one or more heterologous genes. In other cases, the cells are engineered to upregulate expression of one or more genes endogenous to the cells, such as by manipulating one or more regulatory elements of one or more genes endogenous to the cells.

[0068] In certain embodiments, immune cells are engineered to express the BCL6 gene and one or more pro-survival or anti-apoptotic or cell survival-promoting genes (and there may or may not be overlap in the genes classified as pro-survival or anti-apoptotic or cell survival-promoting). As used herein, a pro-survival gene refers to a nucleic acid polymer that can exert an anti-apoptotic function or promote survival by any mechanism. Nucleic acid polymers that can exert an anti-apoptotic function can be one or more Bcl2 family genes (e.g., BCL-xL, BCL-2, MCL-1, Bcl-w, Bfl-1, BCL-B, etc.). Nucleic acid polymers that can exert an anti-apoptotic function can be one or more inhibitor of apoptosis (IAP) family genes (e.g., XIAP, c-IAP1, C-IAP2, NAIP, and survivin, etc.). Nucleic acid polymers capable of exerting anti-apoptotic functions may be able to inhibit or knock out the expression of one or more caspases that play a role in apoptosis (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14). The nucleic acid polymer for knockdown or knockout may be an shRNA expression cassette, or these caspase genes may also be knocked out by gene editing methods (CRISPR, TALEN, zinc finger method, etc.). Nucleic acid polymers capable of exerting anti-apoptotic functions may be able to inhibit or knock out the expression of one or more pro-apoptotic genes (e.g., BIM, Puma, Noxa, Bik, Bmf, Bad, Hrk, Bid, BAX, BAK, BOK, etc.).Nucleic acid polymers capable of exerting anti-apoptotic functions may have anti-apoptotic effects (e.g., insulin-like growth factor (IGF-1), Hsp70, Hsp27, cFLIP, BNIP3, FADD, Akt and NF-κB, Raf-1 and MEK1, p90Rsk, c-Jun, BNIP2, BAG1, HSPA9, HSP90B1, miRNA21, miR-106b-25, miR-206, miR-221 / 222, miR-17-92, miR-133, miR-143, miR-145, miR-155, miR-330, etc.).

[0069] Infinite T cells can be generated using wild-type or mutant BCL6. The present inventors have demonstrated that infinite T cells can be generated using either wild-type or mutant BCL6 that differ in a specific single nucleotide (i.e., the codon for amino acid 395 in wild-type BCL6 is CCT (encoding proline / P), while the codon for amino acid 395 in mutant BCL6 is CTT (encoding leucine / L)). The nucleotide and amino acid sequences of these two BCL6 genes are shown below (the mutation site in the wild-type sequence is underlined).

[0070] Wild-type BCL6 aa sequence: MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCESRAFAPSLYSGLSTPPASYSM YSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEGP EQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPRSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC (SEQ ID NO: 1)

[0071] Nucleotide sequence of wild-type BCL6 (mutation codons in the wild-type sequence are underlined): cCt gagcaggctgagctgggccgcctttccccacgagcctacacggccccacctgcctgccagccacccatggagcctgagaaccttgacctccagtccccaaccaagctgagtgccagcgggggaggactccaccatcccacaagccagccggctcaataacatcgttaacaggtccatgacgggctctccccgcagcagcagcgagagccactcaccactcatcatgcacccccc gaagtgcacgtcctgcggctctcagtccccacagcatgcagagatgtgcctccacaccgctggccccacgttccctgaggagatgggagagaccagtctgagtactcagattctagctgtgagaacggggccttcttctgcaatgagtgtgactgccgcttctctgaggaggcctcactcaagaggcacacgctgcagacccacagtgacaaaccctacaagtgtgaccgct gccaggcctccttccgctacaagggcaacctcgccacacaagaccgtccataccggtgagaaaccctatcgttgcaacatctgtggggcccagttcaacggccagccaacctgaaaacccacactcgaattcactctggagagaagccctacaaatgcgaaacctgcggagccagatttgtacaggtggcccacctccgtgcccatgtgcttatccacactggtgagaagccctatccctgtgaaatctgtggcacccgtttcccggcaccttcagactctgaagagccaccctgcgaatccacacaggagaaaccttaccattgtgagaagtgtaacctgcatttccgtcaaaaagccagctgacttcacttgcgccagaagcatggcgcatcaccaacaccaaggtgcaataccgcgtgtcagccagccagccagaagccatcaccaacaccaaggtcaacaataccgcgtgtcagccagccatgacctgcctccggagctccccaaagccgc (sequence number 2)

[0072] Mutant BCL6 aa sequence (leucine mutations are underlined): MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCESRAFAPSLYSGLSTPPASYSM YSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEG L EQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPRSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC (SEQ ID NO: 3)

[0073] Nucleotide sequence of mutant BCL6 (codon for leucine is underlined):

[0074] cTt gagcaggctgagctgggccgcctttccccacgagcctacacggccccacctgcctgccagccacccatggagcctgagaaccttgacctccagtccccaaccaagctgagtgccagcgggggaggactccaccatcccacaagccagccggctcaataacatcgttaacaggtccatgacgggctctccccgcagcagcagcgagagccactcaccactcatcatgcacccccc gaagtgcacgtcctgcggctctcagtccccacagcatgcagagatgtgcctccacaccgctggccccacgttccctgaggagatgggagagaccagtctgagtactcagattctagctgtgagaacggggccttcttctgcaatgagtgtgactgccgcttctctgaggaggcctcactcaagaggcacacgctgcagacccacagtgacaaaccctacaagtgtgaccgct gccaggcctccttccgctacaagggcaacctcgccacacaagaccgtccataccggtgagaaaccctatcgttgcaacatctgtggggcccagttcaacggccagccaacctgaaaaccacactcgaattcactctggagagaagccctacaaatgcgaaacctgcggagccagatttgtacaggtggcccacctccgtgcccatgtgcttatccacactggtgagaagccctatccctgtgaaatctgtggcacccgtttcccggcaccttcagactctgaagagccaccctgcgaatccacacaggagaaaccttaccattgtgagaagtgtaacctgcatttccgtcaaaaagccagctgacttcacttgcgccagaagcatggcgcatcaccaacaccaaggtgcaataccgcgtgtcagccagccatgacctgcctccggagctccccaaagcctgc (sequence number 4)

[0075] The above immune cells include T cells (e.g., regulatory T cells, CD4 + T cells, CD8 + The immune cells may be any type of immune cell, including T cells, alpha beta T cells, gamma delta T cells, or a mixture thereof, NK cells, invariant NKT cells, NKT cells, innate lymphoid cells, or a mixture thereof. The immune cells may be virus-specific, express a CAR, and / or express a TCR. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells (DCs), mast cells, eosinophils, and / or basophils. Also provided herein are methods for producing and manipulating the immune cells, as well as methods for using and administering the cells for adoptive cell therapy (in which the cells may be autologous or allogeneic). Thus, the immune cells can be used as immunotherapies, such as for targeting cancer cells. The immune cells can be used therapeutically as a single cell type or as a combination of multiple immune cell types. In specific embodiments, the immune cells are CD3+, CD4+, CD8+, CD16+, or a mixture thereof.

[0076] The immune cells can be isolated from a subject, particularly a human subject.These immune cells can be obtained from a subject of interest (for example, a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, or a subject undergoing treatment for a particular disease or condition).Immune cells can be collected from any location in a subject where they exist, including but not limited to blood, umbilical cord blood, spleen, thymus, lymph node and bone marrow.Isolated immune cells can be used directly or can be stored for a certain period of time, such as by freezing.

[0077] The immune cells may be enriched / purified from any tissue in which they reside, including, but not limited to, blood (including blood collected by a blood bank or umbilical cord blood bank), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained via biopsy procedures. The tissues / organs from which immune cells are enriched, isolated, and / or purified can be isolated from both living and non-living subjects. The non-living subject is an organ donor. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some embodiments, immune cells isolated from umbilical cord blood have high immunoregulatory potential, as measured by suppression of CD4+ or CD8+ T cells. In a specific embodiment, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood, for high immunoregulatory potential. Pooled blood can be blood from two or more sources (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more sources (e.g., donor subjects)).

[0078] The population of immune cells can be obtained from a subject in need of treatment or a subject suffering from a disease associated with low immune cell activity. Thus, these cells can be autologous to the subject in need of treatment. Alternatively, the population of immune cells can be obtained from a donor (e.g., a partially or completely histocompatibility-matched donor or a completely histocompatibility-mismatched donor). The immune cell population can be collected from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present in the subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, for example, pooled umbilical cord blood.

[0079] When the immune cell population is obtained from a donor different from the subject, the donor may be allogeneic, provided that the resulting cells are matched to the subject in that the cells can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) matched.

[0080] AT cells In some embodiments, the immune cells are T cells. Several basic approaches for the induction, activation, and expansion of functional anti-tumor effector cells have been reported in the past 20 years. These include autologous cells such as tumor-infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous DCs or PBMCs, lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells isolated by target cell membrane capture; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells that have been genetically reprogrammed or "redirected" to express tumor-reactive or chimeric TCR molecules that exhibit antibody-like tumor recognition capabilities, known as "T-bodies." These approaches have led to numerous protocols for preparing and immunizing T cells, which can be used in the methods described herein.

[0081] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some embodiments, the cells are human cells. The cells are typically primary cells, e.g., cells isolated directly from a subject and / or cells isolated and frozen from a subject. In some embodiments, the cells include one or more subsets of T cells or other cell types (e.g., the entire T cell population, CD4 + cells, CD8 +The present invention includes cells and subpopulations thereof, e.g., subpopulations defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization and / or persistence, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. With respect to the subject being treated, the cells may be allogeneic and / or autologous. In some embodiments, such as with existing technologies, the cells are pluripotent and / or multipotent (e.g., stem cells such as induced pluripotent stem cells (iPSCs)). In some embodiments, the method includes isolating cells from the subject, preparing, treating, culturing, and / or manipulating them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0082] T cells (e.g., CD4 + and / or CD8 + T cell) subtypes and subpopulations include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes (e.g., stem cell memory T (TSC) M ), Central Memory T (TC M ), Effector Memory T(T EM ) or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, intrinsic and adaptive regulatory T (Treg) cells, helper T cells (e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), alpha / beta T cells, and gamma / delta T cells.

[0083] In some embodiments, one or more of the T cell populations are enriched or depleted for cells that are positive for a specific marker, such as a surface marker, or that are negative for a specific marker. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain T cell populations (e.g., non-memory cells), but are present or expressed at relatively high levels on certain other T cell populations (e.g., memory cells).

[0084] In some embodiments, T cells are isolated from PBMC samples by negative selection of markers (e.g., CD14) expressed on non-T cells (e.g., B cells, monocytes or other leukocytes). + or CD8 + By using a selection process, CD4 + Helper T cells and CD8 + Cytotoxic T cells are isolated. + and CD8 + The population can be further sorted into subpopulations by positive or negative selection of markers that are expressed or expressed to a relatively high degree on one or more subpopulations of naive, memory and / or effector T cells.

[0085] In some embodiments, CD8 + The T cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM ) cells or stem cell memory cells are enriched to enhance efficacy, such as improving long-term survival, expansion and / or engraftment following administration, which in some embodiments is particularly robust in such subpopulations.

[0086] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a suspension of single cells is obtained. The single cell suspension is purified in any suitable manner, for example, mechanically (e.g., by gentleMACS). TM Tumors can be disaggregated using a dissociator (Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single cell suspensions of the enzymatic digest of tumors are cultured in interleukin-2 (IL-2) or other growth factors.

[0087] The cultured T cells can be pooled and rapidly expanded. Rapid expansion over about 10 to about 14 days results in an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50, 60, 70, 80, 90, or 100-fold or more). More preferably, rapid expansion over about 10 to about 14 days results in an increase of at least about 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900, or more).

[0088] Expansion can be achieved by any of several methods known in the art. For example, T cells can be readily expanded using nonspecific T cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. The nonspecific T cell receptor stimulation can include approximately 30 ng / ml of OKT3 (available from Ortho-McNeil®, Raritan, NJ), a mouse monoclonal anti-CD3 antibody. Alternatively, T cells can be readily expanded by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more cancer antigens (including antigenic portions thereof, such as epitopes, or cells) in the presence of a T cell growth factor (e.g., 300 IU / ml of IL-2 or IL-15 (IL-2 is preferred)). The cancer antigens can be expressed from a vector, as needed, and are, for example, human leukocyte antigen A2 (HLA-A2)-binding peptides or peptides that bind to other MHC class I or class II molecules. The in vitro-induced T cells are rapidly expanded by restimulation with the same cancer antigen pulsed on antigen-presenting cells expressing HLA-A2 or other HLA molecules. The in vitro-induced T cells can also be expanded in the absence of antigen-presenting cells.

[0089] The autologous T cells can be modified to express T cell growth factors or T cell differentiation factors that promote the growth, differentiation, and activation of the autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, IL-18, IL-21, and IL-12. Suitable modification methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rded., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain embodiments, the modified autologous T cells express high levels of T cell growth factors. T cell growth factor coding sequences (e.g., the coding sequence for IL-12) are readily available in the art, as are promoters whose operably linked sequences promote high level expression of the T cell growth factor coding sequence.

[0090] B.NK cells In some embodiments, the immune cells are natural killer (NK) cells. NK cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against various tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. In humans, NK cells can be detected by specific surface markers such as CD16, CD56, and / or CD8. NK cells do not express T cell antigen receptors, pan-T marker CD3, or surface immunoglobulin B cell receptors.

[0091] In certain embodiments, NK cells are obtained from human peripheral blood mononuclear cells (PBMCs), unstimulated leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, tissue, or umbilical cord blood by methods well known in the art.

[0092] C.NKT cells Natural killer T (NKT) cells are a heterogeneous population of T cells that share characteristics of both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds to self and foreign lipids and glycolipids. They account for only approximately 0.1% of all peripheral blood T cells. NKT cells are a subset of T cells that co-express the αβ T cell receptor but also express various molecular markers typically associated with NK cells, such as NK1.1. Invariant natural killer T (iNKT) cells express high levels of the transcription factor promyelocytic leukemia zinc finger, and their development is dependent on this transcription factor. Currently, five major distinct iNKT cell subsets exist. These subsets produce distinct sets of cytokines upon activation. The subtypes iNKT1, iNKT2, and iNKT17 closely resemble Th cell subsets in cytokine production. Additionally, there are subtypes that specialize in T follicular helper-like and IL-10-dependent regulatory functions.

[0093] D. Innate lymphoid cells Innate lymphoid cells (ILCs) are a group of innate immune cells that originate from common lymphoid progenitors (CLPs) and belong to the lymphoid lineage. These cells are defined by the absence of antigen-specific B or T cell receptors due to the lack of recombination-activating genes (RAGs). ILCs do not express myeloid or dendritic cell markers. Because they play a role in protective immunity and the control of homeostasis and inflammation, their dysregulation can lead to immunopathologies such as allergy, bronchial asthma, and autoimmune diseases. ILCs can be divided based on the cytokines they produce and the transcription factors that control their development and function.

[0094] III. Creation of infinite immune cells In some embodiments, the present disclosure provides a method for extending the lifespan of immune cells by overexpressing BCL6 and one or more pro-survival or anti-apoptotic or cell survival-promoting genes (including one or more anti-apoptotic BCL-2 family genes, such as Bxl-xL). The gene expression can be achieved by conventional molecular biology methods (e.g., cloning the coding sequences of BCL6 and anti-apoptotic BCL-2 family genes downstream of a constitutive or inducible promoter in one or more viral or non-viral vectors and delivering the vectors to the immune cells). Alternatively, the gene expression can be achieved by using CRISPR or other transposases to specifically transcribe mRNA of BCL6 and anti-apoptotic BCL-2 family genes (for example) in the immune cells. The expression of BCL6 and / or anti-apoptotic BCL-2 family members (e.g., Bcl-xL) can be controllable, including by constitutive or inducible means. In some cases, expression of BCL6 and / or an anti-apoptotic BCL-2 family member can have a first type of expression control (e.g., constitutive), and expression of one or more other genes in the system (e.g., genes on the same or another vector) can be controlled in the same manner (e.g., constitutive) or differently (e.g., inducible). In specific cases, BCL6-BCL-xL is controlled by a tet-off or tet-on regulatory mechanism.

[0095] In one exemplary method, the coding sequences of the BCL6 gene and the Bcl-xL gene (for example only) can be linked, but separated by an element that ultimately allows the BCL6 molecule and the Bcl-xL molecule to be produced separately. For example, the coding sequences of the BCL6 gene and the Bcl-xL gene can be linked, but separated by a T2A sequence to create a single open reading frame that can simultaneously express the BCL6 gene and the Bcl-xL gene. This BCL6-T2A-Bcl-xL open reading frame can be cloned into a vector such as a lentiviral vector. The viral vector can then be used to transduce immune cells such as T cells in the presence of, for example, IL-2 and / or IL-15. This method allows the creation of T cell lines, called "infinite T cells," from T cells of healthy donors, that can proliferate in the presence of recombinant human IL-2 and / or IL-15. In some cases, these cells are generated in the presence of IL-2 and / or IL-15 and the cells themselves express heterologous IL-2 and / or IL-15, while in other cases only one of these parameters is used.

[0096] Examples of self-cleaving sequences are:

[0097] T2A (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 5)

[0098] P2A (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 6)

[0099] E2A (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO: 7)

[0100] F2A (GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 8)

[0101] In other cases, an IRES element is used in place of the 2A sequence.

[0102] In some embodiments, the cells are engineered to express the BCL6-2A-BCLxL sequence (SEQ ID NO: 9), which comprises human BCL6, the 2A self-cleaving peptide, and the BCL-xL coding sequence.

[0103]

[0104] Another example of an expression construct containing BCL6 and Bcl-xL is below, where the single underlined part is BCL6, the non-underlined part is P2A, and the double underlined part is Bcl-xL:

[0105] JPEG0007802362000001.jpg220170

[0106] An example of a construct containing BCL6 together with Bcl-xl (L5x(MSCV-BCL6-P2A-BCL-xl-T2A-rtTA); see Figure 21) is below. The general structure is:

[0107] NNNN-CMV promoter NN-HIV-LTR-HIV1_psypak-spacer-RRE-spacer-cPPT-MSCV promoter-BCL-6 WT-P2A-BCL-xL-T2A-rtTA-WPRE-U3PPT-HIV-LTR-bGH pA-SV40 origin of replication-plasmid origin of replication-Ampicin resistance gene-AmpR promoter-NNNN. Specific sequences for particular domains of the constructs below (and in Figure 21) are shown immediately following SEQ ID NO: 11 below:

[0108] GCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAC (SEQ ID NO: 11)

[0109] CMV promoter

[0110] ACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGC (SEQ ID NO: 61)

[0111] HIV LTR

[0112] GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA (SEQ ID NO: 62)

[0113] HIV1 Psi Pack

[0114] TGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAG (SEQ ID NO: 63)

[0115] RRE

[0116] AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGACGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGACAATTTGCTGAGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCT (Sequence number 64)

[0117] cPPT

[0118] AAAAGAAAAGGGGGGA (SEQ ID NO: 65)

[0119] MSCV promoter

[0120] aatgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcggcgcgccagtcctccgatagactgcgtcgcccgggtacccgtattcccaataaagcctcttgctgtttgcatccgaatcgtggactcgctgatccttgggagggtctcctcagattgattgactgcccacctcgggggtctttcat (SEQ ID NO: 66)

[0121] BCL-6 WT

[0122]

[0123] P2A

[0124] GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT (SEQ ID NO: 68)

[0125] BCL-xL

[0126] AGATCTGGAATGTCTCAGAGCAACCGGGAGCTGGTGGTTGACTTTCTCTCCTACAAGCTTTCCCAGAAAGGATACAGCTGGAGTCAGTTTAGTGATGTGGAAGAGAACAGGACTGAGGCCCCAGAAGGGACTGAATCGGAGATGGAGACCCCCAGTGCCATCAATGGCAACCCATCCTGGCACCTGGCAGACAGCCCCGCGGTGAATGGAGCCACTGGCCACAGCAGCAGTTTGGATGCCCGGGAGGTGATCCCCATGGCAGCAGTAAAGCAAGCGCTGAGGGAGGCAGGCGACGAGTTTGAACTGCGGTACCGGCGGGCATTCAGTGACCTGACATCCCAGCTCCACATCACCCCAGGGACAGCATATCAGAGCTTTGAACAGGTAGTGAATGAACTCTTCCGGGATGGGGTAAACTGGGGTCGCATTGTGGCCTTTTTCTCCTTCGGCGGGGCACTGTGCGTGGAAAGCGTAGACAAGGAGATGCAGGTATTGGTGAGTCGGATCGCAGCTTGGATGGCCACTTACCTGAATGACCACCTAGAGCCTTGGATCCAGGAGAACGGCGGCTGGGATACTTTTGTGGAACTCTATGGGAACAATGCAGCAGCCGAGAGCCGAAAGGGCCAGGAACGCTTCAACCGCTGGTTCCTGACGGGCATGACTGTGGCCGGCGTGGTTCTGCTGGGCTCACTCTTCAGTCGGAAA (SEQ ID NO: 69)

[0127] T2A

[0128] GGCAGTggcgagggtagaggttctctcctcacttgtggtgatgttgaagaaaaccctggtcca (SEQ ID NO: 70)

[0129] rtTA

[0130] atgtctagactggacaagagcaaagtcataaacggagctctggaattactcaatggtgtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatgctggacaggcatcatacccacttctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccgctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgagacaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaacagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaaGGTgA (SEQ ID NO: 71)

[0131] WPRE

[0132] TCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCA (SEQ ID NO: 72)

[0133] U3PPT

[0134] AAAAGAAAAGGGGGGA(SEQ ID NO: 73)

[0135] -HIV-LTR

[0136] GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA (SEQ ID NO: 74)

[0137] bGH pA

[0138] CGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATG (SEQ ID NO: 75)

[0139] SV40 replication origin

[0140] Atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcc (SEQ ID NO: 76)

[0141] Plasmid replication origin

[0142] TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA (SEQ ID NO: 77)

[0143] Ampicillin resistance gene

[0144] TTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT (SEQ ID NO: 78)

[0145] AmpR promoter

[0146] ATTGTCTCATGAGCGGATACATATTTGAA (SEQ ID NO: 79)

[0147] In a further aspect, the present disclosure provides infinite immune cells that can be genetically modified to confer favorable properties for targeting specific organ sites or tumor markers. The infinite immune cells can express one or more suicide or elimination genes that can be used to eliminate the infinite immune cells from patients in the event of a serious adverse event. The infinite immune cells can express one or more genes, including genes encoding IL-2 and / or IL-15, which can maintain or enhance the proliferation of infinite T cells for in vivo applications. Expression of IL-2 and / or IL-15 can be constitutive or regulatable (e.g., doxycycline-regulatable (Tet-on or Tet-off)). The cells may express one or more other cytokines (e.g., IL-7, IL-12, IL-18, IL-21, etc.); one or more chemokine receptors (e.g., CCR1, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR7 (ACKR3), CX3CR1, CCRL2 (ACKR5), etc.) and / or one or more other chemokines (e.g., CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CX3CL1, CXCL4L1, etc.

[0148] Infinite immune cells can be engineered to express antigen-specific CARs or TCRs that target tumors or infectious diseases. Another strategy for targeting tumors is to engineer Infinite T cells to express CARs with Fc receptors on their extracellular domains, which can then be used with monoclonal antibodies against tumor markers. Furthermore, Infinite immune cells can be engineered to express specific chemokine receptors and / or adhesion molecules, including integrins, selectins, adhesion molecules belonging to the immunoglobulin superfamily, cadherins, and the CD44 family, to preferentially direct the transport of these cells to the desired organ site.

[0149] Further embodiments provide infinite immune cells with one or more safety switches, such as any type of suicide or elimination gene. In some embodiments, the system may use a truncated human epidermal growth factor receptor (hEGFRt), HSV-TK, SR39-mutated HSV-TK, or the yeast CD gene or its mutant CD20. When hEGFRt is used, this gene can endow the infinite T cells with the property of being recognized and eliminated by FDA-approved monoclonal antibodies, such as cetuximab, when the infinite T cells are no longer needed. For example, this gene can act as a safety switch in the event of a serious adverse event following the injection of therapeutic infinite immune cells. In addition to acting as a safety switch, hEGFRt can also serve as a marker for enriching CAR-positive cells and tracking these cells after infusion into patients.

[0150] An example of a truncated EGFR is the following, in which domains 1 and 2 of EGFR are deleted:

[0151]

[0152] Amino acid sequence of truncated EGFR lacking domains 1 and 2:

[0153] MLLLVTSLLLCELPHPAFLRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR (SEQ ID NO: 13)

[0154] In certain embodiments, the fusion protein as a safety switch is a fusion of EGFR (domain 3) and HER2 (domain IV) fusion protein. In such cases, EGFR domain 3 is the antibody binding domain, and HER2 domain 4 includes the extracellular spacer and transmembrane domain. In a specific embodiment, the fusion protein is a molecule separate from the CAR.

[0155] Any one or more of the genes or expression constructs in the infinite cells may or may not be regulatable, for example, by a Tet-on or Tet-off system, in a doxycycline-regulatable manner. Examples of Tet-responsive promoter sequences include the following Tet-responsive promoters, which contain seven repeats of the Tet-responsive element:

[0156] gagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgatgtcgagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcag tgatagagaacgtatgtcgagtttatccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgtatgtcgaggtaggcgtgtacggtgggaggcctataagcagagctcgtttagtgaaccgtcagatcgcc (SEQ ID NO: 14)

[0157] For the tet system, an example of the DNA sequence for tTA (Tet off) is as follows:

[0158] ATGAGCCGCCTGGATAAGTCCAAAGTGATCAACTCTGCCCTGGAGCTGCTGAATGAAGTGGGCATCGAGGGCCTGACCACACGGAAGCTGGCCCAGAAGCTGGGAGTGGAGCAGCCAACCCTGTACTGGCACGTGAAGAACAAGCGCGCCCTGCTGGACGCCCTGGCCATCGAGATGCTGGATCGGCACCACACACACTTCTGCCCCCTGGAGGGAGAGTCCTGGCAGGATTTCCTGCGGAACAATGCCAAGAGCTTTAGATGTGCACTGCTGTCCCACAGGGACGGAGCAAAGGTGCACCTGGGCACCAGGCCTACAGAGAAGCAGTACGAGACCCTGGAGAACCAGCTGGCCTTCCTGTGCCAGCAGGGCTTTTCTCTGGAGAATGCACTGTATGCACTGAGCGCCGTGGGACACTTCACCCTGGGATGCGTGCTGGAGGACCAGGAGCACCAGGTGGCCAAGGAGGAGAGAGAGACACCCACCACAGATTCCATGCCCCCTCTGCTGAGGCAGGCCATCGAGCTGTTTGACCACCAGGGAGCAGAGCCTGCCTTCCTGTTTGGCCTGGAGCTGATCATCTGCGGCCTGGAGAAGCAGCTGAAGTGTGAGTCTGGAGGACCAGCAGACGCCCTGGACGATTTCGACCTGGATATGCTGCCCGCCGATGCCCTGGACGATTTTGACCTGGATATGCTGCCTGCCGACGCCCTGGACGATCTGGACCTGGATATGCTGCCAGGCacc (SEQ ID NO: 15)

[0159] Examples of the amino acid sequences of tTA (Tet off) are as follows:

[0160] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQG FSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDLDLDMLPG (SEQ ID NO:16)

[0161] An example of the DNA sequence of rtTA (Tet on) is as follows:

[0162] atgtctagactggacaagagcaaagtcataaacggagctctggaattactcaatggtgtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatgctggacaggcatcatacccacttctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccgctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgagacaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaacagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaa (SEQ ID NO: 17)

[0163] Examples of the amino acid sequence of rtTA (Tet on) are as follows:

[0164] MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (SEQ ID NO: 18)

[0165] In some embodiments, the infinite immune cells may be engineered to express one or more cytokines, including IL-2 and / or IL-15 (e.g., inducible IL-2 and / or IL-15), such as to maintain or enhance proliferation. However, in specific cases, any cytokine in the system may be homeostatically controlled. For example, the infinite immune cells may produce IL-15 and / or IL-2 in the presence of an inducer such as doxycycline to support their own proliferation. By adjusting the dosage of doxycycline, the survival and proliferation of the infinite immune cells can be maintained or controlled in vivo.

[0166] Specific IL-2 sequences can be used. In at least some cases, there are two exemplary DNA sequences for IL-2, both of which encode the same IL-2 amino acid sequence.

[0167] IL-2 DNA sequence 1:

[0168] ATGTATCGGATGCAACTCCTCAGCTGCATTGCGTTGTCACTCGCACTCGTCACGAACTCTGCACCGACATCTAGTAGTACTAAGAAAACACAGTTGCAACTGGAGCACCTGCTGTTGGATTTGCAAATGATCCTTAACGGGATCAACAACTACAAAAACCCTAAGCTCACACGAATGCTTACTTTCAAGTTTTACATGCCGAAAAAAGCCACAGAGCTGAAGCATCTTCAGTGCCTTGAAGAGGAGCTTAAACCCCTCGAGGAGGTACTGAATCTCGCGCAAAGCAAGAATTTTCATTTGCGGCCCCGGGACCTTATATCAAACATTAACGTGATCGTGTTGGAACTCAAGGGATCAGAGACGACATTTATGTGCGAGTACGCTGACGAGACCGCTACAATCGTAGAGTTTCTCAATAGGTGGATCACGTTTTGCCAAAGCATCATCTCAACGCTC (SEQ ID NO: 19)

[0169] DNA sequence 2 of IL-2:

[0170] ATGTATAGGATGCAGCTGCTGTCCTGCATCGCCTTGTCCCTGGCCCTTGTGACCAACAGCGCCCAACCTCCTCCTCTACCAAAAAAACCCAACTTCAGCTTGAGCATCTCCTCTTGGACCTGCAGATGATCCTGAATGGTATAAACAACTACAAGAACCCCAAGCTGACCCGGATGCTTACATTCAAATTCTATATGCCTAAAAAGGCTACAGAGCTGAAGCACCTGCAG TGCCTGGAAGAGGAGCTGAAGCCACTGGAAGAGGTCCTGAACTTGGCCCAGAGCAAGAACTTTCACCTCAGGCCCAGGGACTTGATAAGCAACATAAATGTAATCGTCCTGGAGCTGAAGGGTCTGAAACAACCTTCATGTGTGAGTATGCAGATGAGACCGCTACCATCGTGGAGTTCCTCAACAGATGGATTACATTTTGTCAATCCATCATCAGCACCCTGACATCT (Sequence number 20)

[0171] In certain embodiments, a specific IL-2 amino acid sequence is used in the cells:

[0172] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL (SEQ ID NO: 21)

[0173] In certain embodiments, a specific IL-15 nucleic acid polymer sequence is used in the cells:

[0174] ATGGGCCTGACCTCTCAGCTGCTGCCACCCCTGTTCTTTCTGCTGGCCTGTGCCGGCAATTTCGTGCACGGCGCCAACTGGGTGAATGTGATCTCTGACCTGAAGAAGATCGAGGATCTGATCCAGAGCATGCACATCGACGCCACCCTGTATACAGAGTCCGATGTGCACCCTTCTTGCAAGGTGACAGCCATGAAGTGTTTTCTGCTG GAGCTGCAGGTCATCTCTCTGGAGAGCGGCGACGCCAGCATCCACGATACCGTGGAGAATCTGATCATCCTGGCCAACAATAGCCTGAGCTCCAACGGCAATGTGACAGAGTCCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCCTTTGTGCACATCGTGCAGATGTTTATCAATACCTCTTGA (Sequence number 22)

[0175] In certain embodiments, a specific IL-15 amino acid sequence is used in the cells:

[0176] MGLTSQLLPPLFFLLACAGNFVHGANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 23)

[0177] In certain cases, the immune cells comprise IL-15 fused to part or all of the IL-15 receptor. In specific cases, the immune cells comprise IL-15 fused to the sushi domain of the IL-15 receptor alpha unit, an example of which is the following sequence:

[0178] MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 24)

[0179] DNA sequence of IL-15 fused to the sushi domain of the IL-15 receptor alpha unit:

[0180] ATGGCACCTAGAAGAGCCAGAGGATGTAGAACACTGGGGACTGCCAGCGCTCCTTCTTTTGTTGCTGCTGAGACCACCTGCAACTCGCGGAATCACTTGTCCTCCTCTATGAGTGTGGAACACGCTGACATTTGGGTCAAGTCCTACTCTCTGTATTCCCGGGAGAGATATATATGTAACTCTGGTTTCCAAACGCAAGGCAGGCACCAGCAGCCTTACCGAGTGTGCTTAACAAGGCAAATGTGGCTCACTGGACAACACCTTCTCTGAAGTGCATGAGATGGAGGGGAGGATCAGGTGGAGGAGGTTCTGGTGGGGGGGATCAAATT GGGTGAACGTAATTTCCGACCTGAAAAAGATCGAAGATCTCCATTCAAAGCATGCATATCGATGCCACCCTCCTATACCGAGAGCGATGTCCACCCATCCTGCAAAGTTACGGCGATGAAATGCTTCCTGCTCGAGCTCCAGGTTATTTCTCTGGAGAGCGGGATGCCTCCATCCCACGATACTGTCGAGAACCTCATATTCTGGCCAATAACTCCCTGTCTAGCAATGGCAATGTGACTGAATCAGGTTGCAAGGAGTGCGAGGAGCTCGAAGAGAAAAACATAAAAGAATTCCTGCAATCCTTTGTCCCATATCGTACAGATGTTTTATCAACACAGCAGC (sequence number 25)

[0181] Infinite immune cells can be genetically engineered to confer target selectivity to the infinite immune cells by introducing one or more chimeric antigen receptors (CARs) capable of recognizing specific tumor markers, such as CD19, CD20, CD22, and / or mesothelin; and / or T cell receptors (TCRs), such as those for EBV, CMV, or NY-ESO-1. One example is the "anti-CD19 infinite CAR T cells" (CD19 inCART) referred to elsewhere herein. CD19 is expressed in almost all types of B-cell lymphoma or leukemia and normal B cells. CD19 in CART is produced by delivering a lentiviral or non-viral vector expressing an anti-CD19 CAR into selected infinite immune cells.

[0182] Infinite immune cells can also be genetically engineered to confer additional properties, such as i) resistance to T cell exhaustion by knocking out or knocking down inhibitory receptors or ligands such as PD-1, LAG-3, TIM-3, and PD-L1, ii) resistance to immunosuppressive mechanisms by knocking out or knocking down TGF-β receptors, iii) prevention of graft-versus-host disease by knocking out TCRs, iv) improved efficacy by expressing surface or intracellular molecules such as cytokines or cytotoxic molecules, and v) improved in vivo persistence by making them resistant to elimination by host immune cells, including T cells and NK cells. This can be achieved by knocking out or knocking down MHC molecules or expressing surface ligands or other surface or intracellular molecules on the Infinite immune cells to suppress or reduce host immune cell function.

[0183] Infinite immune cells can be generated by specific methods or under specific conditions. For example, in specific embodiments, the infinite immune cells being generated can be subjected to one or more specific agents during their generation, which agents increase the efficacy of the cells at the time of generation, at least compared to the efficacy in the absence of exposure. For example, in some cases, IL-2 is used to generate and expand infinite T cells. In specific embodiments, one or more different combinations of cytokines (such as IL-2, IL-7, IL-21, IL-15, IL-12, IL-18, IL-23, IFN-gamma, TNF-alpha, etc.) and / or chemokines can be used to prepare infinite T cells with specific phenotypes and functions.

[0184] IV. Genetically Engineered Antigen Receptors The immune cells of the present disclosure may or may not be genetically engineered to express one or more antigen receptors (e.g., one or more engineered TCRs and / or one or more CARs). For example, the immune cells may be modified to express a CAR and / or TCR with antigen specificity for a cancer antigen or a microbial antigen (including a pathogenic antigen). Multiple CARs and / or TCRs (e.g., for various antigens) may be added to the immune cells. In some embodiments, the immune cells are engineered to express a CAR or TCR by knocking in the CAR or TCR into the locus of an inhibitory gene using gene editing methods such as CRISPR / Cas9.

[0185] Suitable modification methods are known in the art. See, e.g., Sambrook and Ausubel, supra. For example, the cells can be transduced to express a TCR with antigen specificity for a cancer antigen using the transduction methods described in Heemskerk et al., 2008 and Johnson et al., 2009.

[0186] As an option to overcome the long-term problem of autoreactivity caused by pairing of retrovirally transduced TCR chains with endogenous TCR chains, electroporation of RNA encoding full-length TCR α and β (or γ and δ) chains can be used. Even if such alternative pairing occurs in transient transfection strategies, the transduced TCR α and β chains are only transiently expressed, so any autoreactive T cells that may be generated lose this autoreactivity after a period of time. When expression of the transduced TCR α and β chains declines, only normal autologous T cells remain. This is not the case when full-length TCR chains are introduced by stable retroviral transduction; the transduced TCR chains are not lost, resulting in the constant presence of autoreactivity in the patient.

[0187] In some embodiments, the cells contain one or more nucleic acid polymers introduced via genetic engineering that encode one or more antigen receptors, and the genetically engineered products of such nucleic acid polymers. In some embodiments, the nucleic acid polymers are heterologous, i.e., not normally present in the cell or sample obtained from the cell (e.g., obtained from another organism or cells that are not normally found, e.g., in the cell being engineered and / or the organism from which such cells are derived). In some embodiments, the nucleic acid polymers are not naturally occurring, such as nucleic acid polymers not found in nature (e.g., chimeras).

[0188] In some embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to one or more antigens. In some embodiments, the antigen is a protein, lipid, or carbohydrate expressed on the surface of a cell, including certain cancer cells. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen (e.g., a peptide antigen of an intracellular protein) that is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule, similar to a TCR.

[0189] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering and introducing these receptors into cells are described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. 2002131960, 2013287748, and 2013 / 123061. Nos. 130149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, and European Patent Application No. EP 2537416, and / or Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some embodiments, the genetically engineered antigen receptor includes a CAR, such as that described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO / 2014055668A1.

[0190] A. Chimeric antigen receptor In some embodiments, the CAR comprises a) an intracellular signaling domain, b) a transmembrane domain, c) an extracellular domain comprising an antigen-binding region, and optionally d) one or more costimulatory domains.

[0191] In some embodiments, engineered antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). These CARs generally comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some embodiments via a linker and / or transmembrane domain. Such molecules typically mimic or mimic signaling through a natural antigen receptor, through such a receptor in conjunction with a costimulatory receptor, and / or through a costimulatory receptor alone.

[0192] Certain embodiments of the present disclosure relate to the use of nucleic acid polymers that include nucleic acid polymers encoding antigen-specific CAR polypeptides (including humanized CARs (hCARs) to reduce immunogenicity) that include an intracellular signaling domain, a transmembrane domain, and an extracellular domain containing one or more signaling motifs. In certain embodiments, the CAR can recognize an epitope that includes a space shared between one or more antigens. In certain embodiments, the binding region can include the complementarity-determining region of a monoclonal antibody, the variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide that binds to a receptor (e.g., a cytokine).

[0193] It is contemplated that the human CAR nucleic acid polymer may be a human gene used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present invention comprises a full-length cDNA or coding region of a CAR. The antigen-binding region or domain is the V of a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody. H Chain and V LThe fragments may include fragments of the chains (e.g., those described in U.S. Patent No. 7,109,304, incorporated herein by reference). The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0194] The configuration can be a multimer (e.g., a diabody or multimer). The multimer is most likely formed by cross-pairing of the variable portions of the light and heavy chains into a diabody. The hinge portion of the construct can have several options, ranging from a complete deletion, to maintaining the first cysteine, to substituting proline instead of serine, to truncating up to the first cysteine. The Fc portion can be deleted. Any stable and / or dimerizing protein can serve this purpose. Only one of the Fc domains can be used, for example, the CH2 or CH3 domain of a human immunoglobulin. The hinge, CH2, and CH3 regions of a human immunoglobulin modified to improve dimerization can also be used. Only the hinge portion of an immunoglobulin can also be used. A portion of CD8 alpha or a synthetic molecule can also be used.

[0195] In some embodiments, the CAR nucleic acid comprises partial or complete sequences encoding other costimulatory receptors (e.g., the native or modified extracellular, transmembrane, and intracellular signaling domains of particular molecules such as CD28), alone or in combination. Other costimulatory domains include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), ICOS, HVEM, GITR, LIGHT, CD40L, DR3, CD30, SLAM, CD2, CD226 (DNAM-1), MyD88, CD244, TMIGD2, BTNL3, NKG2D, DAP10, DAP12, 4-1BB (CD137), or synthetic molecules. In addition to the primary signal elicited by CD3ζ, additional signals provided by the costimulatory receptor inserted into the CAR are important for the full activation of NK cells and may help improve in vivo persistence and therapeutic success of adoptive immunotherapy.

[0196] In some embodiments, CARs are constructed to have specificity for a particular antigen (or marker or ligand) (e.g., an antigen expressed in a particular cell type targeted by adoptive therapy (e.g., a cancer marker) and / or an antigen intended to induce an attenuated response (e.g., an antigen expressed on a normal or non-diseased cell type)). Thus, the CAR typically comprises one or more antigen-binding molecules (e.g., one or more antigen-binding fragments, domains, or portions) or one or more antibody variable domains, and / or antibody molecules in its extracellular portion. In some embodiments, the CAR comprises the antigen-binding portion of an antibody molecule (e.g., a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb)).

[0197] In certain embodiments of chimeric antigen receptors, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain containing the antigen-binding region) comprises a tumor-associated antigen or a pathogen-specific antigen-binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors such as Dectin-1. The tumor-associated antigen may be of any type, as long as it is expressed on the cell surface of tumor cells. Exemplary tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, and the like. In certain embodiments, the CAR can be coexpressed with cytokines to improve persistence when the amount of tumor-associated antigen is low. For example, the CAR can be coexpressed with IL-15.

[0198] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA, cDNA, or synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been found to stabilize mRNA. It may also be beneficial to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0199] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector.Methods for stably transfecting cells by electroporation using naked DNA are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding the chimeric receptor that is contained in a plasmid expression vector in the appropriate orientation for expression.

[0200] Alternatively, viral vectors (such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors or lentiviral vectors) can be used to introduce chimeric constructs into immune cells.The vector suitable for use according to the method of the present disclosure is a non-replicating vector in immune cells.There are many known virus-based vectors (such as HIV, SV40, EBV, HSV or BPV-based vectors), and the copy number of the virus that is maintained in cells is low enough to maintain the viability of the cells.

[0201] In some embodiments, the antigen-specific binding component or antigen-specific recognition component is linked to one or more transmembrane domains and intracellular signaling domains.In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR.In one embodiment, the transmembrane domain that naturally associates with one of the domains in the CAR is used.In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane protein, in order to minimize interaction with other members of the receptor complex.

[0202] In some embodiments, the transmembrane domain is derived from natural or synthetic sources. If natural in origin, the domain is derived from any membrane-bound or transmembrane protein in some embodiments. The transmembrane region includes a transmembrane region derived from (i.e., including at least the transmembrane region of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD2, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8 (including CD8 alpha), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, PD-1, CTLA4, and DAP molecules. Alternatively, the transmembrane domain is a synthetic transmembrane domain in some embodiments. In some embodiments, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine, hi some embodiments, triplets of phenylalanine, tryptophan and valine may be found at each end of the synthetic transmembrane domain.

[0203] The hinge region of the CAR may be located at the N-terminal end of the transmembrane domain, and in some embodiments, it is derived from natural or synthetic origin.The hinge sequence may also be referred to as spacer or extracellular spacer, and is generally the extracellular structural region of the CAR that separates the binding unit from the transmembrane domain.In certain embodiments, the CAR comprises an immunoglobulin (Ig)-like domain hinge.The hinge generally provides stability for efficient CAR expression and activity. The hinge may be derived from any suitable source, but in specific embodiments, the hinge is derived from CD8a, CD28, PD-1, CTLA4, the alpha, beta, or zeta chain of the T-cell receptor, CD2, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8b, CD9, CD16, CD22, CD27, CD32, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD160, BTLA, LAIR1, TIGIT, TIM4, ICOS / CD278, GITR / CD357, NKG2D, LAG-3, PD-L1, PD-1, TIM-3, HVEM, LIGHT, DR3, CD30, CD224, CD244, SLAM, CD226, DAP, or a combination thereof.

[0204] In certain embodiments, the platform technologies disclosed herein for genetically modifying immune cells, such as T cells or NK cells, include: (i) non-viral gene transfer using an electroporation device (e.g., nucleofector); (ii) CARs that signal through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations); (iii) CARs with an extracellular domain of variable length that connects the antigen recognition domain to the cell surface; and, in some cases, (iv) CARs + and artificial antigen-presenting cells (aAPCs) derived from K562, which can robustly and numerically expand immune cells ( Singh et al., 2008 ; Singh et al., 2011 ).

[0205] In certain embodiments, the cells are engineered to express a CD19-CAR sequence (SEQ ID NO: 26) comprising the VH and VL of an anti-CD19 antibody, a fusion sequence of a CD8 hinge (any hinge may be referred to as a spacer or extracellular spacer) and transmembrane domain, and signaling domains of CD3 and CD28.

[0206]

[0207] A specific example of a CAR (FMC63-CD8a hinge / TM-CD28-CD3z) that can be used is as follows:

[0208] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 27)

[0209] FMC63-CD8a Hinge / TM-CD28-CD3z

[0210] An example of an anti-CD19 CAR comprising anti-CD19 scFv FMC63, the hinge and transmembrane domain of CD8a, the CD28 costimulatory domain, and CD3 zeta (FMC63-CD8a hinge / TM-CD28-CD3z) is as follows:

[0211] In the example of SEQ ID NO: 28, the following components of the CAR are delimited as follows: CD8 signal peptide ATGGCCCTGCCAGTGACCGCCCTGCTGCTGCCACTGGCACTGCTGCTGCACGCAGCAAGGCCA (SEQ ID NO: 29) FMC63 light chain GACATCCAGATGACACAGACCACAAGCTCCCTGTCCGCCTCTCTGGGCGACAGAGTGACCATCTCTTGCAGGGCCAGCCAGGATATCTCCAAGTATCTGAATTGGTACCAGCAGAAGCCTGATGGCACAGTGAAGCTGCTGATCTATCACACCTCTAGAC TGCACAGCGGCGTGCCATCCAGGTTTAGCGGCTCCGGCTCTGGCACAGACTACTCTCTGACCATCAGCAATCTGGAGCAGGAGGATATCGCCACCTATTTCTGCCAGCAGGGCAACACACTGCCTTACACCTTTGGCGGCGGCACAAAGCTGGAGATCACC (Sequence number 30) Linker GGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCC (SEQ ID NO: 31) heavy chain GAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCCAGCCAGTCCCTGTCTGTGACATGTACCGTGTCCGGCGTGTCTCTGCCAGACTACGGCGTGAGCTGGATCAGACAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCCGAGACCACATACTATAACTCCGCCCTGAAGTCTCGGCTGACCATCATCAAGGACAACAGCAAGTCCCAGGTGTTTCTGAAGATGAATTCCCTGCAGACAGACGATACCGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCTTATGCCATGGATTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGC (SEQ ID NO: 32) CD8a hinge ACCACAACCCCTGCACCAAGACCACCAACACCAGCACCTACCATCGCAAGCCAGCCTCTGTCCCTGAGGCCAGAGGCATGCAGGCCAGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTCGCCTGCGAT (SEQ ID NO: 33) CD8TM ATCTACATCTGGGCACCACTGGCAGGAACATGTGGAGTGCTGCTGCTGTCTCTGGTCATCACCCTGTATTGTTGGGTG (SEQ ID NO: 34) CD28 co-stimulatory domain AGAAGCAAGAGATCCAGGCTGCTGCACAGCGACTACATGAATATGACACCAAGGAGACCAGGACCAACCAGGAAGCACTATCAGCCTTACGCACCTCCAAGGGACTTCGCAGCATATAGGAGC (SEQ ID NO: 35) CD3 zeta AGGGTGAAGTTTTCTCGCAGCGCCGATGCCCCAGCCTATcAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGAGGAGAGGAAGGGATCCAGAGATGGGAGGCAAGCCTAGGCGCAAGAACCCACAGGAG GGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGAGAGAGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGTCCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCACTGCCACCTAGA (Sequence number 36)

[0212] The corresponding amino acid sequence of FMC63-CD8a hinge / TM-CD28-CD3z is as follows: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 37)

[0213] In the example of SEQ ID NO: 37, the following components of the CAR are delimited as follows: CD8 signal peptide MALPVTALLLPLALLLHAARP (SEQ ID NO: 38) FMC63 light chain JPEG0007802362000002.jpg11170 (CDR is in bold) (SEQ ID NO: 39) Linker GGGGSGGGGSGGGGS (SEQ ID NO: 40) heavy chain JPEG0007802362000003.jpg16170 (bold characters are CDRs) (SEQ ID NO: 41) CD8a hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 42) CD8™ IYIWAPLAGTCGVLLLSLVITLYCWV (SEQ ID NO: 43) CD28 costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 44) CD3 Zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 45)

[0214] FMC63-CD28 Hinge / TM-CD28-CD3z

[0215] An example of an anti-CD19 CAR comprising anti-CD19 scFv FMC63, the hinge and transmembrane domain of CD28, the CD28 costimulatory domain, and CD3 zeta (FMC63-CD28hinge / TM-CD28-CD3z) is as follows: The amino acid sequence of FMC63-CD28hinge / TM-CD28-CD3z is as follows: MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAK HYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 47) Another example of a nucleic acid sequence for an FMC63-CD28 Hinge-TM CAR is as follows: CD28 Hinge: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 49) CD28 hinge nucleic acid sequence ATCGAAGTGATGTATCCACCCCCTTACCTGGATAACGAGAAGAGCAATGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCATCTCCCCTGTTCCCTGGCCCAAGCAAGCCC (SEQ ID NO: 50) CD28™ domain FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 51)

[0216] FMC63-PD-1 Hinge-TM CAR An example of a CAR with the following components is: CSF2RA signal peptide-FMC63 light chain-linker-heavy chain-PD1 hinge-PD-1TM-CD28 costimulatory-CD3 zeta: MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNT LPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNS LQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSQVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVIERSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 52) The nucleic acid sequence of the FMC63-PD-1 Hinge-TM CAR is as follows: PD-1 hinge QVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 54) PD-1 TM domain VGVVGGLLGSLVLLVWVLAVI (SEQ ID NO: 55)

[0217] FMC63-CTLA4 Hinge-TM CAR: CSF2RA signal peptide-FMC63 light chain-linker-heavy chain-CTLA4 hinge-CTLA-4 TM-CD28 costimulatory-CD3 zeta MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG NTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSvidpepcpdsdfllwilaavssglffysflltaRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 56) CSF2RA signal peptide MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 58) CTLA4 hinge VIDPEPCPDSD (SEQ ID NO: 59) CTLA4 TM domain FLLWILAAVSSGLFFYSFLLT (SEQ ID NO: 60)

[0218] BT cell receptor (TCR) In some embodiments, the genetically engineered antigen receptor includes a recombinant TCR and / or a TCR cloned from a naturally occurring T cell. "T cell receptor" or "TCR" refers to a molecule comprising a variable α chain and a variable β chain (also known as TCRα and TCRβ, respectively) or a variable γ chain and a variable δ chain (also known as TCRγ and TCRδ, respectively), which can specifically bind to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type. In alternative embodiments, the cell lacks an engineered TCR; for example, an endogenous TCR within the cell can target cancer or an infectious disease (e.g., a CMV- or EBV-specific T cell with an endogenous TCR).

[0219] TCRs, which typically exist as αβ and γδ types, are generally similar in structure, although the T cells expressing them may differ in anatomical location or function. TCRs may be found on the cell surface or in soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes) and are typically involved in recognizing antigens bound to major histocompatibility complex (MHC) molecules on the surface. In some embodiments, TCRs may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., 1997). For example, in some embodiments, each chain of a TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, TCRs associate with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs of the αβ or γδ types.

[0220] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment (e.g., an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e., an MHC-peptide complex). An "antigen-binding portion" or antigen-binding fragment of a TCR, which may be used interchangeably, refers to a molecule that includes only a portion of the structural domain of the TCR but binds to the antigen (e.g., an MHC-peptide complex) that the complete TCR binds. In some cases, the antigen-binding portion includes sufficient variable domains of the TCR (e.g., the variable a chain and variable β chain of the TCR) to form a binding site for binding to a specific MHC-peptide complex, e.g., each chain typically includes three complementarity-determining regions.

[0221] In some embodiments, the variable domains of TCR chains associate to form loops, or complementarity-determining regions (CDRs) similar to immunoglobulins, which mediate antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. Typically, as in immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the primary CDR involved in recognizing processed antigens, although CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, whereas CDR1 of the beta chain interacts with the C-terminal portion of the peptide. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the beta chain may contain an additional hypervariable (HV4) region.

[0222] In some embodiments, a TCR chain comprises a constant domain. For example, similar to an immunoglobulin, the extracellular portion of a TCR chain (e.g., a chain, β chain) comprises two immunoglobulin domains: a variable domain (e.g., V) at the N-terminus; a or Vp; usually Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest," US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed., amino acids 1-116), and one constant domain adjacent to the cell membrane (e.g., the a-chain constant domain or C aThe TCR may comprise a α-chain constant domain or Cp, typically amino acids 117-259 according to Kabat, and a β-chain constant domain or Cp, typically amino acids 117-295 according to Kabat. For example, in some cases, the extracellular portion of the TCR formed by the two chains comprises two membrane-proximal constant domains and two membrane-distal variable domains containing the CDRs. The constant domain of the TCR domain contains a short connective sequence in which cysteine ​​residues form disulfide bonds, thereby forming a link between the two chains. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α-chain and β-chain such that the TCR contains two disulfide bonds in the constant domains.

[0223] In some embodiments, the TCR chain may include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail. In some cases, due to its structure, the TCR can associate with other molecules such as CD3. For example, a TCR that includes a constant domain along with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0224] Generally, CD3 is a multiprotein complex that can have three distinct chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, this complex can contain a CD3γ chain, a CD3δ chain, two CD3ε chains, and a homodimeric CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a property that allows these chains to associate with positively charged T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), while each CD3ζ chain contains three. Generally, ITAMs are involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in transmitting signals from the TCR to the cell. The CD3 chain and the ζ chain associate with the TCR to form a complex known as the T cell receptor complex.

[0225] In some embodiments, the TCR can be a heterodimer of two chains, α and β (or optionally γ and δ), or can be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α and β or γ and δ) linked, such as by a disulfide bond. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, a nucleic acid polymer encoding the TCR can be obtained from various sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, e.g., a cell, e.g., a T cell (e.g., a cytotoxic T cell), T cell hybridoma, or other publicly available source. In some embodiments, the T cell can be obtained from an in vivo isolated cell. In some embodiments, a high-affinity T cell clone can be isolated from a patient and the TCR isolated. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, the TCR clone against the target antigen is a clone produced in a transgenic mouse engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, for example, tumor antigens (e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against the target antigen (see, for example, Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR, or antigen-binding portion thereof, can be synthetically generated with knowledge of the sequence of the TCR.

[0226] C. Antigen-presenting cells Antigen-presenting cells, including macrophages, B lymphocytes, and dendritic cells, are distinguished by the expression of specific MHC molecules. APCs internalize antigens and re-express portions of the antigen along with MHC molecules on the outer membrane of their cell membrane. The MHC is a large genetic complex containing multiple loci. MHC loci encode two major classes of MHC membrane molecules, termed class I and class II MHC. T helper lymphocytes generally recognize antigens associated with MHC class II molecules, while T cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. The MHC is referred to as the HLA complex in humans and the H-2 complex in mice.

[0227] In some cases, aAPC is useful when preparing the therapeutic compositions and cell therapy products of the above-mentioned embodiments.For the general guidance of the preparation and use of antigen-presenting system, see, for example, United States Patent No. 6,225,042, United States Patent No. 6,355,479, United States Patent No. 6,362,001 and United States Patent No. 6,790,662; United States Patent Application Publication No. 2009 / 0017000 and United States Patent Application Publication No. 2009 / 0004142; and International Publication No. WO2007 / 103009.

[0228] The aAPC system may include at least one exogenous assisting molecule. Any suitable number and combination of assisting molecules may be used. The assisting molecules may be selected from assisting molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include CD86, CD64 (FcγRI), 41BB ligand, and IL-21. Adhesion molecules may include, for example, carbohydrate-binding glycoproteins such as selectins, transmembrane glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecules (ICAMs), which promote cell-cell or cell-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs such as ICAM-1. Techniques, methods, and reagents useful for the selection, cloning, preparation, and expression of exemplary assisting molecules, including costimulatory molecules and adhesion molecules, are exemplified, for example, in U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

[0229] D. Antigen Antigens targeted by genetically engineered antigen receptors or naturally expressed antigen receptors (e.g., TCRs) on immune cells include antigens expressed in the context of diseases, conditions, or cell types targeted via adoptive cell therapy. These diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematological cancers, cancers of the immune system (e.g., lymphomas, leukemias, and / or myelomas, e.g., B, T, and myeloid leukemias, lymphomas, and multiple myeloma). In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., tumor cells or pathogenic cells, compared to normal cells or tissues or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0230] Any suitable antigen can be used in this method.Exemplary antigens include, but are not limited to, antigenic molecules derived from infectious agents, autoantigens / self-antigens, tumor-associated antigens / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015).In certain embodiments, these antigens include CD19, CD20, CD22, CD30, CD70, CD79a, CD79b, SLAM-F7NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, CEA. In certain embodiments, antigens for one or more antigen receptors include, but are not limited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4 and / or CEA. Sequences for these antigens are known in the art, for example: CD19 (accession number NG_007275.1), EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11), NY-ESO (accession number NC_000023.11), EGFRvIII (accession number NG_007726.3), MUC1 (accession number NG_029383.1), HER2 ...HER2 (accession number NG_007275.1), HER2 (accession number NG_007275.1), HER2 (accession number NG_007275.1), HER2 (accession number NG_007275.1), HER2 (accession number NG_007275.1), HER2 (accession number NG_007275.1), HER2 (accession number NG_007275.1), HER2 (accession number NG_007275.1), HER2 (accession number NG_007275.1), HER2 (accession number NG_007275.1), HER2 (accession number 2 (accession number NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12), and / or CEA (accession number NC_000019.10).

[0231] Tumor-associated antigens can be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma, ovarian cancer or melanoma. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3 and MAGE4 (or other MAGE antigens, for example, those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma and bladder cancer. For example, see U.S. Patent No. 6,544,518. Tumor-associated antigens of prostate cancer include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1 and six-transmembrane epithelial antigen of the prostate (STEAP).

[0232] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, tumor antigens can be self-peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH), a short 10-amino acid peptide useful in the treatment of many cancers.

[0233] Tumor antigens include tumor antigens derived from cancers characterized by the expression of tumor-associated antigens, such as HER-2 / neu expression. Tumor-associated antigens of interest include lineage-specific tumor antigens, such as the melanocyte-melanoma lineage antigen MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase, and tyrosinase-related proteins. Exemplary tumor-associated antigens include p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CEA, CDK-4 / m, ELF2M, GnT-V, G250 , HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling transduction complex 1 (TACSTD1), TACSTD2, receptor tyrosine kinase (TKI) For example, epidermal growth factor receptor (EGFR) (particularly EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinase (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription factors STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-kappa B (NF-B),Notch receptors (e.g., Notch1-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAG These include, but are not limited to, tumor antigens derived from or including one or more of E1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and idiotypes.

[0234] Antigens can include epitope regions or epitope peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells (e.g., aberrantly expressed intronic sequences such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and N-acetylglucosaminyltransferase-V); clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens containing epitope regions or epitope peptides derived from oncoviral processes (e.g., human papillomavirus proteins E6 and E7); Epstein-Barr virus protein LMP2; and tumor-selectively expressed, non-mutated oncofetal proteins (e.g., carcinoembryonic antigen and alpha-fetoprotein).

[0235] In certain embodiments, the antigen may be an antigen of a microorganism. In some embodiments, the antigen is obtained or derived from a pathogenic microorganism or an opportunistic pathogenic microorganism (also referred to herein as an infectious disease microorganism) (e.g., a virus, a fungus, a parasite, and a bacterium). In certain embodiments, the antigen derived from such a microorganism includes a full-length protein.

[0236] Illustrative pathogenic organisms having antigens contemplated for use in the methods described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B, and C, vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and JC virus), adenovirus, coronaviruses such as SARS-CoV, SARS-CoV-2, or MERS, Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species, including Streptococcus pneumoniae. As one of skill in the art will appreciate, proteins from these and other pathogenic microorganisms for use as antigens as described herein, as well as the nucleotide sequences encoding those proteins, can be identified in publications and public databases such as GENBANK®, SWISS-PROT® and TREMBL®.

[0237] Antigens derived from human immunodeficiency virus (HIV) include HIV virion structural proteins (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or any of the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0238] Antigens derived from herpes simplex viruses (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. The late group of genes primarily encodes proteins that form virion particles. Such proteins include the five proteins that form the viral capsid (UL): UL6, UL18, UL35, UL38, and the major capsid proteins UL19, UL45, and UL27, each of which can be used as an antigen as described herein. Other exemplary HSV proteins contemplated for use as antigens herein include ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD) proteins. The HSV genome contains at least 74 genes, each of which encodes a protein that can potentially be used as an antigen.

[0239] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed during immediate early and early stages of viral replication, glycoproteins I and III, capsid protein, coat protein, lower matrix protein pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products of the UL128-UL150 gene cluster (Rykman, et al., 2006), envelope glycoprotein B (gB), gH, gN, and pp150. As one of skill in the art will appreciate, CMV proteins for use as antigens described herein can be identified in public databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see, e.g., Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[0240] Antigens derived from Epstein-Van virus (EBV) contemplated for use in certain embodiments include EBV proteins produced during the latent infection cycle, including EBV lytic proteins gp350 and gp110, Epstein-Van nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), and latent membrane protein (LMP)-1, LMP-2A, and LMP-2B (see, e.g., Lockey et al., 2008).

[0241] Antigens derived from respiratory syncytial virus (RSV) contemplated for use herein include any of 11 proteins or antigenic fragments thereof encoded by the RSV genome: NS1, NS2, N (nucleocapsid protein), M (matrix protein) SH, G and F (viral coat proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcriptional control), RNA polymerase, and phosphoprotein P.

[0242] Antigens derived from vesicular stomatitis virus (VSV) contemplated for use include any one of the five major proteins and their antigenic fragments encoded by the VSV genome: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, e.g., Rieder et al., 1999).

[0243] Antigens derived from influenza virus contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[0244] Exemplary viral antigens include adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigens), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (hepatitis B core or surface antigen, hepatitis C virus E1 or E2 glycoproteins, core or nonstructural proteins), herpesvirus polypeptides (including herpes simplex virus or varicella zoster virus glycoproteins), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, and the like. Also included are, but are not limited to, polypeptides of viruses, orthomyxoviruses, papillomaviruses, parainfluenza virus polypeptides (e.g., hemagglutinin polypeptides and neuraminidase polypeptides), paramyxoviruses, parvoviruses, pestiviruses, picornaviruses (e.g., poliovirus capsid polypeptides), poxviruses (e.g., vaccinia virus polypeptides), rabies virus polypeptides (e.g., rabies virus glycoprotein G), reoviruses, retroviruses, and rotaviruses.

[0245] In certain embodiments, the antigen can be a bacterial antigen. In certain embodiments, the bacterial antigen of interest can be a secreted polypeptide. In certain other embodiments, the bacterial antigen includes an antigen that has a portion of the polypeptide exposed on the extracellular surface of the bacterium.

[0246] Antigens from Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), that are intended for use include virulence regulators, such as Agr system, Sar and Sae, Arl system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), Srr system and TRAP.Other Staphylococcus proteins that can serve as antigens include Clp protein, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, for example, Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes of two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example, at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As one of skill in the art will appreciate, Staphylococcus proteins for use as antigens can also be identified in other public databases, such as GenBank®, Swiss-Prot®, and TrEMBL®.

[0247] Antigens derived from Streptococcus pneumoniae contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as antigens in some embodiments (see, e.g., Zysk et al., 2000). The entire genome sequence of virulent strains of Streptococcus pneumoniae has been sequenced, and as one of skill in the art will appreciate, S. pneumoniae proteins for use herein may also be identified in other public databases, such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest as antigens according to the present disclosure include virulence factors and proteins predicted to be exposed on the surface of pneumococcus (see, for example, Frolet et al., 2010).

[0248] Examples of bacterial antigens that can be used as antigens include Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae OspA), b-type outer membrane proteins), Helicobacter polypeptides, Klebsiella polypeptides, L-form bacteria polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacterium polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Streptococcus pneumoniae polypeptides (i.e., S.pn eumoniae polypeptides) (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rosalimea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, Group A Streptococcus polypeptides (e.g., M protein of S. pyogenes), Group B Streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., F1 and V antigens of Y. pestis).

[0249] Examples of fungal antigens include Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucorum polypeptides, and Pelomyces polypeptides. These include, but are not limited to, polypeptides of the genus Psilomyces, polypeptides of the genus Penicillium, polypeptides of the genus Phialemonium, polypeptides of the genus Phialophora, polypeptides of the genus Prototheca, polypeptides of the genus Pseudoalescheria, polypeptides of the genus Pseudomicrodochium, polypeptides of the genus Phytium, polypeptides of the genus Rhinosporidium, polypeptides of the genus Rhizopus, polypeptides of the genus Scolecobasidium, polypeptides of the genus Sporothrix, polypeptides of the genus Stemphylium, polypeptides of the genus Trichophyton, polypeptides of the genus Trichosporon, and polypeptides of the genus Xylohypha.

[0250] Examples of protozoan parasite antigens include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, and Plasmodium polypeptides. Examples of helminth parasite antigens include Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothria polypeptides, Dipridium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Ancylostoma polypeptides, Nematogyrus polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascaris polypeptides, Physaroptera polypeptides,Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides, Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichuris polypeptides, Trichuria polypeptides, Uncinaria polypeptides, and Uchleria polypeptides (e.g., P. falciparum circumsporozoite polypeptide (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSA1) c-terminus), and export protein 1 (PfExp-1), Pneumocystis polypeptide, Sarcocystis polypeptide, Schistosoma polypeptide, Theileria polypeptide, Toxoplasma polypeptide, and Trypanosoma polypeptide.

[0251] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens and allergens) of fleas; ticks, including hard mites and ulcerative mites; flies, such as midges, mosquitoes, sand flies, black flies, bot flies, horn flies, deer flies, tsetse flies, stable flies, flies that cause myiasis and small biting gnats; ants; spiders, lice; mites; and hemipteran insects, such as bed bugs and assassin bugs.

[0252] E. Suicide gene The infinite immune cells of the present disclosure (including immune cells that can express one or more CARs and / or one or more engineered TCRs) can contain one or more suicide genes. As used herein, the term "suicide gene" is defined as a gene whose gene product is converted into a compound that kills host cells when a prodrug is administered. Examples of suicide gene / prodrug combinations that can be used include truncated EGFR and cetuximab; herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside.

[0253] V. Methods of Delivery to the Cells Those skilled in the art would be fully capable of constructing vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) to express the antigen receptors of the present disclosure. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors (including replication-competent, replication-deficient, and gutless forms thereof), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, Maraba virus vectors, and the like. These include, but are not limited to, adenovirus (A virus) vectors and group B adenovirus enadenotucirev vectors.

[0254] A. Viral Vectors Viral vectors encoding BCL6 and cell survival-promoting genes and / or antigen receptors may be provided in certain embodiments of the present disclosure. When creating recombinant viral vectors, non-essential genes are usually replaced with genes or coding sequences for heterologous (or non-native) proteins. Viral vectors are a type of expression construct that utilizes viral sequences to introduce nucleic acid polymers and, in some cases, proteins into cells. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into the genome of host cells and stably and efficiently express viral genes, makes these viruses attractive candidates for transferring foreign nucleic acid polymers into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acid polymers in certain embodiments of the present disclosure are described below.

[0255] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994,136).

[0256] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and nucleic acid polymer sequence expression both in vivo and ex vivo.For example, recombinant lentiviruses that can infect non-dividing cells (wherein suitable host cells are transfected with two or more vectors that have packaging functions, namely gag, pol and env, and rev and tat) are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference.

[0257] B. Regulatory Elements The expression cassette contained in the vector useful in the present disclosure includes, inter alia, a eukaryotic transcription promoter operably linked to a protein-coding sequence, a splice signal including an intervening sequence, and a transcription termination / polyadenylation sequence (5' to 3' direction). The promoters and enhancers that control the transcription of protein-coding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery can collect and integrate the regulatory information carried by each element, allowing different genes to exert different, often complex, patterns of transcriptional control. Promoters used in the context of the present disclosure include constitutive promoters, inducible promoters, and tissue-specific promoters.

[0258] C. Promoter / Enhancer The expression constructs provided herein include a promoter that drives expression of an antigen receptor. Promoters generally contain sequences that function to locate the start site for RNA synthesis. The most well-known example of this is the TATA box, although some promoters, such as the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, lack a TATA box and instead have discrete elements overlapping the start site itself that help fix the location of initiation. Additional promoter elements control the frequency of transcription initiation. These are usually located in the region 30-110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site. To place a coding sequence "under the control of" a promoter, the 5' end of the transcription initiation site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA and promotes expression of the encoded RNA.

[0259] The spacing between promoter elements is often variable, and promoter function is preserved even when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decrease. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0260] A promoter may be a promoter naturally associated with a nucleic acid sequence, such as may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as an "endogenous" promoter. Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not naturally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not naturally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are not "naturally occurring," i.e., contain various elements of various transcriptional control regions and / or expression-altering mutations. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to synthetically producing promoter and enhancer nucleic acid sequences, recombinant cloning and / or PCR TMNucleic acid amplification techniques, including but not limited to, can be used to generate sequences in connection with the compositions disclosed herein. Additionally, it is contemplated that regulatory sequences that direct transcription and / or expression of sequences in organelles other than the nucleus (e.g., mitochondria, chloroplasts, etc.) can be used as well.

[0261] Naturally, it will be important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are aware that a combination of promoter, enhancer, and cell type is generally used for protein expression (see, e.g., Sambrook et al. 1989, incorporated herein by reference). The promoter used can be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter that directs high-level expression of the introduced DNA segment and is useful under appropriate conditions (e.g., a promoter useful in the large-scale production of recombinant proteins and / or recombinant peptides). The promoter can be heterologous or endogenous.

[0262] Additionally, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Data Base EPDB via the World Wide Web at epd.isb-sib.ch / ) can be used to drive expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another viable embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if an appropriate bacterial polymerase is provided as part of the delivery complex or as an additional genetic expression construct.

[0263] Non-limiting examples of promoters include early or late viral promoters (e.g., SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter); eukaryotic promoters (e.g., beta-actin promoter, GADPH promoter, metallothionein promoter); and chain-like response element promoters (e.g., cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA), and minimal TATA box proximal response element promoter (tre)). Human growth hormone promoter sequences (e.g., human growth hormone minimal promoter listed in GenBank, Accession No. X05244, nucleotides 283-341) or mouse mammary tumor promoter (available from ATCC, Cat. No. ATCC 45007) can also be used. In certain embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter; however, any other promoter useful for driving expression of therapeutic genes is also applicable to the practice of the present disclosure.

[0264] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and have the ability to act in cis and in any orientation over relatively long distances (up to several kilobases away from the target promoter).However, enhancer function is not necessarily limited to such long distances, as enhancers can also function proximal to a given promoter.

[0265] D. Initiation Signals and Linked Expression Specific initiation signals may also be used in the expression constructs provided herein for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals (including the ATG initiation codon) may need to be provided. One of ordinary skill in the art would be readily able to determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in-frame" with the reading frame of the desired coding sequence. The exogenous translational control signals and initiation codons may be natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcriptional enhancer elements.

[0266] In certain embodiments, the use of internal ribosome entry site (IRES) elements is used to generate multigene messages, i.e., polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at internal sites. IRES elements from two members of the picornaviridae family (polio and encephalomyocarditis) and IRESs from mammalian messages have been reported. IRES elements can link heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together, generating polycistronic messages. IRES elements allow each open reading frame to be accessible to ribosomes for efficient translation. Multiple genes can also be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0267] Furthermore, certain 2A sequence elements can be used to achieve linked or simultaneous expression of genes within the constructs provided herein. For example, a cleavage sequence can be used to link open reading frames to form a single cistron, thereby co-expressing genes. An exemplary cleavage sequence is F2A (foot-and-mouth disease virus 2A) or a "2A-like" sequence (e.g., Thosea asigna virus 2A; T2A).

[0268] E. Replication starting point To propagate a vector in a host cell, the vector may contain one or more origins of replication (often referred to as "ori"), such as a nucleic acid sequence corresponding to the EBV oriP described above, which is a specific nucleic acid sequence at which replication is initiated, or a genetically engineered oriP with a similar or enhanced function in programming. Alternatively, origins of replication of other viruses that replicate extrachromosomally, as described above, or autonomously replicating sequences (ARS), can be used.

[0269] F. Selectable and Screenable Markers In some embodiments, cells containing the constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker causes an identifiable change in the cell that allows cells containing the expression vector to be easily identified. Generally, a selectable marker is a marker that confers a selectable characteristic. A positive selectable marker is a marker whose presence allows its selection, and a negative selectable marker is a marker whose presence prevents selection. An example of a positive selectable marker is a drug resistance marker.

[0270] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of conditions, other types of markers are contemplated, including colorimetrically based screenable markers such as GFP. Alternatively, screenable enzymes can be used as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immunological markers, perhaps in conjunction with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection markers and screenable markers are well known to those skilled in the art.

[0271] G. Nucleic Acid Polymer Delivery Methods Engineered immune cells can be constructed using any of many established gene transfer methods known to those skilled in the art.In certain embodiments, engineered cells are constructed using viral vector-based gene transfer methods to introduce nucleic acid polymers.Viral vector-based gene transfer methods can include lentiviral vectors, retroviral vectors, adenoviral vectors, or adeno-associated viral vectors.In certain embodiments, engineered cells are constructed using non-viral vector-based gene transfer methods to introduce nucleic acid polymers.In certain embodiments, non-viral vector-based gene transfer methods include gene editing methods selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) nucleases. In certain embodiments, non-viral vector-based gene editing methods include transfection or transformation methods selected from the group consisting of lipofection, nucleofection, virosomes, liposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA uptake.

[0272] The cells can be engineered to express genes of interest and / or antigen receptors by random or site-specific insertion (e.g., by gene editing methods including, but not limited to, meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and CRISPR-Cas systems).

[0273] In addition to viral delivery of nucleic acid polymers encoding genes of interest and / or antigen receptors, the following methods are considered in the present disclosure as additional methods for recombinant gene delivery into a given host cell. The introduction of nucleic acid polymers, such as DNA or RNA, into the immune cells of the present disclosure can be carried out using any method suitable for delivering nucleic acid polymers to transform cells, as described herein or known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA (e.g., ex vivo transfection, injection (including microinjection)); electroporation; calcium phosphate precipitation; the use of DEAE-dextran followed by polyethylene glycol; direct sonication; liposome-mediated transfection and receptor-mediated transfection; microprojectile bombardment; agitation with silicon carbide fibers; Agrobacterium-mediated transformation; desiccation / inhibition-mediated DNA uptake, and any combination of such methods. By applying methods such as these, organelles, cells, tissues, or organisms can be stably or transiently transformed.

[0274] VI. Treatment Method The infinite immune cells can be used in both therapy and research. The infinite immune cells, including T cells or NK cells expressing CAR and / or engineered TCR, can be used to treat cancer, infectious diseases, immune disorders or inflammatory disorders.

[0275] In one method, allogeneic universal CAR T cells targeting antigens such as CD19, CD20, CD22, CD79a, CD79b, or BAFF-R can be used alone or in combination to treat B-cell leukemia and lymphoma. Allogeneic universal anti-mesothelin CAR T cells can be used, for example, to treat mesothelioma, pancreatic adenocarcinoma, or ovarian cancer. NY-ESO-targeted TCR-T cells can be used, for example, to treat melanoma or multiple myeloma. Virus-specific T cells against viruses such as EBV, CMV, BK virus, etc. can be used to treat the respective viral infections. Allogeneic inhibitory or regulatory T cells can be used to treat autoimmune disorders, GVHD, and other inflammatory disorders.

[0276] Gamma / delta T cells and virus-specific T cells are less likely to cause GVHD and, in specific embodiments, provide additional anti-tumor and / or anti-viral functions. In specific embodiments, virus-specific infinite T cells can be used for at least two purposes. First, virus-specific infinite T cells can be used to treat certain viral infections (e.g., CMV or EBV infection) or certain cancers. A second embodiment involves transducing one or more CARs and / or engineered TCRs into virus-specific T cells. Such infinite CAR T cells with endogenous virus-specific TCRs may have potential advantages, such as a lower likelihood of causing GVHD. Cells with such endogenous virus-specific TCRs do not require gene editing methods to knock out the TCR in the T cells. When combining gene editing technologies such as CRISPR / Cas9, virus-specific T cells are not necessarily required to generate CAR T cells. Alternatively, gamma / delta-infinite CAR T cells or CAR-NK or CAR-NKT or CAR-innate lymphoid cells could be used, which do not cause GvHD and are not expected to require TCR knockout.

[0277] When the modified cell lines of the present invention are intended for use in humans, they are first tested for tumoricidal activity and therapeutic efficacy in animal models, such as the NSG mouse model commonly used in cancer research. Such mouse studies are preclinical studies that can be performed before attempting therapeutic use in patients.

[0278] Infinite immune cells can be used to treat cancers, including hematological and non-hematological malignancies, by administering to patients effective amounts of engineered cytotoxic Infinite T cells expressing various CARs or TCRs against various tumor targets, either alone or in combination. For example, to treat patients with B-cell leukemia or lymphoma, CD19inCART (one of which is Ie1-L4aJ3 cells (CD8-positive cells from healthy donor 1 transduced with a CAR against human CD19 with a truncated human EFGR marker)) can be administered together with IL-2 or IL-15. The Ie1-L4aJ3 cells can be present in a conventional pharmaceutical excipient, such as water or buffered saline. When administered to patients, the engineered cells can stop tumor growth by CD19-specific killing. In human patients, the immune cells can be administered by intravenous infusion (iv). However, other administration methods, such as subcutaneous (sc) injection, can also be used. Once the neoplastic cells have been successfully eradicated, these immune cells can be eliminated by withdrawing IL-2 or IL-15 or by injecting anti-EGFR antibodies.

[0279] The appropriate dosage of the Infinite Immune Cells (and one or more cytokines, e.g., IL-2 and / or IL-15, when used) will vary depending on the recipient's age, health, sex, and weight, as well as any other concurrent treatments the recipient is undergoing for related or unrelated conditions. Those skilled in the art can readily determine the appropriate dose of modified cells and drugs to administer to a patient depending on the factors described above. The number of cells that constitutes a tumoricidal effective amount can be determined using animal models. These parameters can be readily determined by those skilled in the art.

[0280] The effectiveness of this treatment against tumors can be determined by detecting any viable tumor cells in the patient's peripheral blood or bone marrow samples, or by other imaging diagnostics such as CT, MRI or PET scans.Similarly, any remaining unwanted modified infinite T cells can be monitored using methods such as flow cytometry and polymerase chain reaction.

[0281] Compared with previous cytotoxic cell lines such as TALL-104 and NK-92 cells, the Infinite Immune Cells are generated from normal immune cells. Therefore, the risk of leukemogenesis associated with the Infinite Immune Cells is lower than that of TALL-104 and NK-92, since the Infinite Immune Cells are not expected to harbor any other unknown tumorigenic gene mutations. Furthermore, the proliferation of the Infinite Immune Cells can be stopped by withdrawing IL-2 or IL-15 administration. This is an unparalleled safety advantage over the leukemia-derived cell lines TALL-104 and NK-92.

[0282] In some embodiments, the present disclosure provides a method for immunotherapy, comprising administering an effective amount of the immune cells of the present disclosure. In certain embodiments of the present disclosure, cancer or infectious diseases are treated by transferring an immune cell population that induces an immune response. Provided herein is a method for treating cancer or delaying the progression of cancer in an individual, comprising administering an effective amount of antigen-specific cell therapy to the individual. The method can be applied to the treatment of immune disorders, solid cancers, blood cancers, and viral infections.

[0283] The tumors for which this treatment method is useful include any malignant cell type, such as those found in solid tumors or blood tumors.Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary blood tumors include bone marrow tumors, T- or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer (including digestive cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0284] The cancer may specifically be cancer of the following histological types, but is not limited to: neoplasia, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular adenocarcinoma Follicular carcinoma; Follicular adenocarcinoma; Papillary-follicular adenocarcinoma; Non-encapsulated sclerosing carcinoma; Adrenal cortical carcinoma; Endometrioid carcinoma; Adnexal carcinoma; Apocrine adenocarcinoma; Sebaceous gland carcinoma; Ceruminous adenocarcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Theca cell tumor, malignant; Granulosa cell tumor, malignant; Androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma Tumor, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Glomus angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Lentigo maligna melanoma; Acral lentiginous melanoma; Nodular melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Mixed Müllerian tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Fetal Cancer; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Paracortical osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillar astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal;Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Lateral granuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin's lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate-grade / follicular NHL; Intermediate-grade diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cleaved cell NHL; Bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0285] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need thereof (e.g., an individual with cancer or an infectious disease). These cells then strengthen the individual's immune system to attack the respective cancer cells or pathogenic cells. In some cases, the individual is provided with immune cells more than once. When the individual is provided with immune cells more than once, the time between administrations should be sufficient for propagation in the individual, and in specific embodiments, the time between administrations is 1, 2, 3, 4, 5, 6, 7 days or more.

[0286] Certain embodiments of the present disclosure provide a method for treating or preventing immune-mediated disorders. In one embodiment, the subject has an autoimmune disease. Non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac polydermatitis, and the like. spate-dermatitis), chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (e.g., microvascular neuropathy), These include: inflammatory bowel disease (e.g., inflammatory bowel disease, focal glomerulosclerosis, or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff man syndrome, systemic lupus erythematosus, ulcerative colitis, uveitis, vasculitis (e.g., polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis, or dermatitis herpetiformis vasculitis), vitiligo, and Wegener's granulomatosis. Therefore, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis or psoriasis.Subjects may also have allergic disorders such as asthma.

[0287] In yet another embodiment, the subject is a recipient of a transplanted organ or stem cells, and immune cells are used to prevent and / or treat rejection. In certain embodiments, the subject has or is at risk of developing graft-versus-host disease. GVHD is a potential complication of any transplant that uses or includes stem cells from a related or unrelated donor. There are two types of GVHD: acute and chronic. Acute GVHD appears within the first three months after transplant. Signs of acute GVHD include a reddish rash on the hands and feet, which may spread with peeling or blistering of the skin and become more severe. Acute GVHD can also affect the stomach and intestines, causing muscle cramps, nausea, and diarrhea. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD is affecting the liver. Chronic GVHD is graded based on its severity: stage / grade 1 is mild; stage / grade 4 is severe. Chronic GVHD develops three months or later after transplantation. The symptoms of chronic GVHD are similar to those of acute GVHD, but chronic GVHD can also affect the mucous glands of the eyes, the salivary glands of the mouth, and the glands that lubricate the stomach wall and intestines. Any immune cell population disclosed herein can be used. Examples of transplanted organs include organ grafts, such as kidney, liver, skin, pancreas, lung, and / or heart, or cell grafts, such as islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The graft can be a composite graft, such as facial tissue. Immune cells can be administered before, simultaneously with, or after transplantation. In some embodiments, the immune cells are administered prior to transplantation, e.g., at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior to transplantation. In one specific, non-limiting example, administration of a therapeutically effective amount of immune cells occurs 3-5 days prior to transplantation.

[0288] In some embodiments, the subject may be administered non-myeloablative lymphodepleting chemotherapy prior to immune cell therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable such treatment, administered by any suitable route. The non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, particularly when the cancer is melanoma, which may be metastatic. An exemplary administration route for cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain embodiments, approximately 60 mg / kg of cyclophosphamide is administered for two days, followed by approximately 25 mg / m 2 of fludarabine is administered for 5 days.

[0289] In certain embodiments, a growth factor or differentiation factor that promotes the proliferation, differentiation, and activation of immune cells is administered to a subject simultaneously with or subsequent to the administration of immune cells. The immune cell growth factor can be any suitable growth factor that promotes the proliferation and activation of immune cells. Examples of suitable immune cell growth or differentiation factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations (e.g., IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7, and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2).

[0290] Therapeutically effective amounts of immune cells can be administered by several routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, intraventricular, intrathecal or intraarticular injection or infusion.

[0291] The therapeutically effective amount of immune cells used in adoptive cell therapy is the amount that achieves the desired effect in the treated subject.For example, this may be the amount of immune cells required to inhibit the progression, or the amount of immune cells required to reverse autoimmune disease or alloimmune disease, or the amount that can relieve symptoms caused by autoimmune disease, such as pain and inflammation.This may be the amount required to relieve symptoms associated with inflammation, such as pain, edema, and elevated body temperature.This may also be the amount required to reduce or prevent rejection of transplanted organs.

[0292] The immune cell population can be administered in a treatment regimen consistent with the disease, for example, once or several times over one to several days, to ameliorate the disease state, or can be administered periodically over a long period of time to inhibit disease progression and prevent disease recurrence. The exact dose used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the physician and each patient's circumstances. The therapeutically effective amount of immune cells depends on the subject being treated, the severity and type of affliction, and the mode of administration. In some embodiments, the dose that can be used in treating human subjects is at least 3.8 x 10 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 or at least 3.8×10 10 immune cells / m 2 In certain embodiments, the dose used in treating a human subject is about 3.8 x 10 9 ~Approx. 3.8×10 10 immune cells / m 2 In a further embodiment, the therapeutically effective amount of immune cells is in the range of about 5×10 6 cells / kg body weight~approx. 7.5×10 8 cells / kg body weight, e.g., about 2 x 10 7 Cells ~ approx. 5 x 108 cells / kg body weight or approximately 5 x 10 7 cells ~ approx. 2 x 10 8 The amount of immune cells can vary depending on the cell / kg body weight.The exact amount of immune cells can be easily determined by those skilled in the art based on the age, weight, sex and physiological condition of the subject.Effective amount can be extrapolated from the dose-response curve derived from in vitro model or animal model test system.

[0293] The immune cells can be administered in combination with one or more other therapeutic agents for treating immune-mediated disorders. Combination therapy can include one or more antibacterial agents (e.g., antibiotics, antiviral agents, and antifungal agents), antitumor agents (e.g., monoclonal antibodies such as rituximab, trastuzumab, etc., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunodepleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine, glucocorticoids, For example, dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids (e.g., hydrocortisone, dexamethasone, or prednisone) or nonsteroidal anti-inflammatory agents (e.g., acetylsalicylic acid, ibuprofen, or naproxen sodium)), cytokines (e.g., interleukin-10 or transforming growth factor-beta), hormones (e.g., estrogen), or vaccines. In addition, immunosuppressants or immune tolerance inducers may be administered, including but not limited to calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., antibodies that recognize CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); irradiation; or chemokines, interleukins, or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents may be administered before, during, or after the administration of immune cells, depending on the desired effect. The administration of the cells and the agent may be by the same route or different routes, and at the same or different sites.

[0294] A. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising infinite immune cells (eg, T cells or NK cells) and a pharmaceutically acceptable carrier.

[0295] Pharmaceutical compositions and formulations as described herein comprise the active ingredient (e.g., antibody or polypeptide) having a desired degree of purity in one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22 ndThe pharmaceutical composition may be prepared in the form of a lyophilized formulation or an aqueous solution by mixing with (Illegible, 2012 edition). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers (e.g., phosphate, citric acid, and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol alcohol, butyl alcohol, or benzyl alcohol; alkyl parabens (e.g., methyl paraben or propyl paraben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues). polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., polyethylene glycol (PEG)). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as neutral active soluble hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is used in combination with one or more additional glycosaminoglycanases, such as chondroitinases.

[0296] B. Combination Therapy In certain embodiments, the compositions and methods of this embodiment comprise an immune cell population that is used in combination with at least one additional treatment.The additional treatment can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, targeted therapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof.The additional treatment can be in the form of adjuvant therapy or neoadjuvant therapy.

[0297] In some embodiments, the additional treatment is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional treatment is administration of a side effect limiting agent (e.g., an agent aimed at reducing the incidence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional treatment may be one or more chemotherapeutic agents known in the art.

[0298] Immune cell therapy can be administered before, during, after, or in various combinations with an additional cancer treatment, such as immune checkpoint therapy. These administrations can occur at intervals ranging from simultaneous administration to minutes, days, or weeks. In embodiments in which immune cell therapy is provided to a patient separately from an additional therapeutic agent, it is common to ensure that no significant time elapses between delivery times so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is contemplated that the antibody therapy and anticancer therapy may be provided to a patient within about 12 to 24 or 72 hours of each other, more particularly, within about 6 to 12 hours of each other. In some situations, if several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between administrations, it may be desirable to significantly extend the treatment period.

[0299] Various combinations can be used. In the example below, immune cell therapy is "A" and anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0300] Administration of any compound or treatment of the present embodiments to a patient follows typical protocols for administering such compounds, taking into account any toxicities of those agents. Thus, in some embodiments, there is a step of monitoring for toxicities that may result from the combination therapy.

[0301] 1.Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within cells, for example, whether they affect the cell cycle and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0302] Examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclosphosphamide); alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa); ethyleneimines and methylamelamines (altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine); acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatins; kallistatins; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleuthecins eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine) rine, prednimustine, trofosfamide, and uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine); antibiotics (e.g., enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11)); dynemicins (including dynemicin A); bisphosphonates (e.g., clodronate); esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autarubicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomicinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g. mitomycin C), mycophenolic acid, nogalarnicin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, pteropterin, and trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprin e) and thioguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine); androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone); anti-adrenals (e.g., mitotane and trilostane); folic acid supplements (e.g., frolinic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;Bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids (e.g., maytansin and ansamitocins); mitoguazone; mitoxantrone; mopidanmol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid); 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecines (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes (e.g., cisplatin, oxaliplatin, and carboplatin); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids (e.g., retinoic acid); capecitabine;Carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein tranferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0303] 2. Radiation therapy Other widely used agents that cause DNA damage include gamma rays, X-rays, and / or what are commonly known as directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents, such as microwaves, proton beam irradiation, and UV irradiation, are also contemplated. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Radioisotope dose ranges vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0304] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapies can be used in conjunction with or in conjunction with the methods and compositions of the present disclosure. In the context of cancer treatment, immunotherapeutics typically rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody can function alone as a therapeutic effector or can recruit other cells to actually affect cell killing. The antibody can also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector can be a lymphocyte bearing a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells, NKT cells, innate lymphoid cells, and NK cells.

[0305] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) covalently linked to cytotoxic drugs and can be used in combination therapy. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver their payload (drug) to tumor cells bearing abundant levels of the antigen. Targeted delivery of the drug also minimizes exposure to normal tissues, resulting in reduced toxicity and an improved therapeutic index. Exemplary ADC drugs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).

[0306] In one aspect of immunotherapy, tumor cells must have some marker that is amenable to targeting, i.e., some marker that is not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, and gamma-IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

[0307] Examples of immunotherapies include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapy such as interferon α, β, and γ, IL-1, GM-CSF, and TNF; gene therapy such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185. It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0308] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either strengthen or weaken signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0309] The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant ligand or receptor, or may be an antibody, particularly a human antibody. Known inhibitors of immune checkpoint proteins or their analogs may be used, particularly chimeric, humanized, or human antibodies. As those skilled in the art will recognize, alternative and / or equivalent names may be used for certain antibodies described in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0310] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand-binding partner. In a specific embodiment, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits PDL1 from binding to its binding partner. In a specific embodiment, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits PDL2 from binding to its binding partner. In a specific embodiment, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0311] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular or PD-1-binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody that may be used. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.

[0312] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 resembles the T cell costimulatory protein CD28; both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also found on regulatory T cells and may be important for the function of these cells. Activation of T cells via the T cell receptor and CD28 leads to high expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0313] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human, humanized, or chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0314] Anti-human CTLA-4 antibodies (or VH and / or VL domains therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or heavy and light chain VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to the same epitope on CTLA-4 as the above-mentioned antibodies and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to the above-described antibody (eg, at least about 90%, 95%, or 99% variable region identity to ipilimumab).

[0315] 4.Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, which physically removes, excises, and / or destroys all or part of the cancerous tissue, and may be used in conjunction with other treatments (e.g., the treatment of the present embodiments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies). Lumpectomy refers to the physical removal of at least part of the tumor. In addition to lumpectomy, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0316] When removing part or all of cancerous cells, tissue or tumor, cavity can be formed in the body.Treatment can be achieved by perfusion, direct injection or local application of additional anti-cancer therapy to the area.Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months.These treatments can also be treatments with various dosages.

[0317] 5. Other agents It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions may enhance the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiating agents may be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis (e.g., antibody c225) may be used in combination with certain aspects of the present embodiments to improve the efficacy of treatment.

[0318] VII. Products or Kits Also provided herein are articles of manufacture or kits containing the unlimited immune cells. The articles of manufacture or kits may further include a package insert containing instructions for using the immune cells to treat or delay the progression of cancer in an individual or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein may be included in the articles of manufacture or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation and a label, which may be attached to or associated with the container and indicate usage. The articles of manufacture or kits may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. In some embodiments, the articles of manufacture further include one or more additional agents (e.g., chemotherapeutic agents and anti-cancer agents). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes. [Example]

[0319] IV. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be recognized by those of skill in the art that the procedures disclosed in the examples which follow are procedures discovered by the inventors to work well in the practice of the invention and can therefore be considered preferred modes for its practice. However, those of skill in the art, in light of the present disclosure, should recognize that many changes can be made in the specific embodiments which are disclosed and which still yield like or similar results without departing from the spirit and scope of the invention.

[0320] Example 1 - Infinite immune cells for adoptive therapy 293T cells were cultured and passaged in 10 mL of high-glucose DMEM medium containing 10% FBS and 1% Pen / Strep in a T75 flask. The next day, when 293T cells reached 90% confluency, they were used for transfection for lentiviral vector generation and plasmid packaging. By linking the coding sequences of the BCL6 and Bcl-xL genes with a T2A sequence, a single open reading frame capable of simultaneous expression of the BCL6 and Bcl-xL genes was created. This BCL6-T2A-Bcl-xL open reading frame was cloned into a lentiviral vector using Gibson assembly according to the protocol provided by NEB. The final vector was designated pLV4a plasmid (Figure 1A). This pLV4a plasmid was co-transfected into 293T cells with the lentiviral vector packaging mix from ABM. The viral supernatant was concentrated using a Clontech Lenti-X concentrator.

[0321] To develop an unlimited cell line derived from a healthy donor, normal T cells were cultured in a RosetteSep™ system manufactured by STEMCELL Technologies. TM Human T Cell Enrichment Cocktail and SepMate TM T cells were isolated from healthy donors using 10-50 tubes. The isolated T cells were then cultured in a 10% FBS, 2% HEPES, 1% sodium pyruvate, and 0.01% 2-mercaptoethanol solution, as well as 50-1000 IU / mL IL-2 (Genscript) and 25 µL / mL ImmunoCult TMT cells were cultured in RPMI-1640 medium (Gibco) supplemented with human CD3 / CD28 / CD2 T cell activator (STEMCELL Technologies). After 36–48 hours of culture, 1 million cultured T cells were transduced with concentrated pLV4a lentiviral vector (Figure 1A) in the presence of RetroNectin (Clontech). The T cells were then cultured in RPMI-1640 medium in the presence of 50–1000 IU / mL IL-2, and passaged and split as needed. Some transduced T cells continued to proliferate indefinitely. Using this method, T cell lines termed "indefinite T cells" were generated from healthy donor T cells, which proliferate in the presence of recombinant human IL-2 or IL-15.

[0322] Next, we used the above method to generate several novel infinite T cell lines. These were designated In1-L4a T cells, consisting of multiple T cell subsets. A series of T cells were isolated and generated by cell sorting or genetic engineering using In1-L4a T cells, including Ie1-L4a, If1-L4a, In1-L4aJ3, Ie1-L4aJ3, Igd1-L4a, and Igd1-L4aJ3. A detailed description of these IL-2- or IL-15-dependent infinite T cell lines is summarized in Table 1. [Table 1]

[0323] In1-L4a cells and derivatives were treated with GlutaMAX TMThey are easily maintained in standard culture media, such as RPMI 1640 medium containing supplements, sodium pyruvate, and 10% fetal bovine serum (FBS). Additionally, 50–1000 IU / mL of recombinant human IL-2 is added for long-term growth (Figure 1B). IL-15 also supported proliferation, but IL-7 or IL-21 did not (Figure 1B). When maintained in suspension culture with twice-weekly media changes, the cells were able to grow and expand very rapidly in an exponential pattern, with a doubling time of approximately 24 hours. When these indeterminate T cells were maintained in culture, they continued to proliferate for over three months in the presence of IL-2 without any change in proliferation rate (Figure 1B).

[0324] These cells were highly dependent on IL-2 for their survival and proliferation, and after IL-2 was removed from the culture medium, they ceased proliferation and rapidly died (Figure 1B). Infinite T cells were CD3 positive, and even after long-term in vitro culture and expansion, other surface markers (e.g., CD4 or CD8, TCRαβ or TCRgδ, or CD16) were expressed on several infinite T cell subsets (Figure 1C). These markers indicated that infinite T cells were a mixed population of different T cell subsets (Figure 1C). Therefore, specific T cell populations could be isolated by cell sorting using specific T cell markers. For example, CD8+ infinite T cells were isolated by cell sorting using an anti-CD8 antibody. Another specific T cell population, γδ T cell population, was also isolated by cell sorting using an anti-TCRgδ antibody. After sorting, a relatively pure γδ T cell line was generated (Figure 1D).

[0325] Mature T cells can further differentiate into distinct functional subsets in lymphoid tissues, such as Th1, Th2, Th17, Treg, and Tfh. Differentiation into these functional subsets is driven by unique master transcription factors. For example, Th1 differentiation is driven by Tbet, Th2 differentiation by GATA-3, Th17 differentiation by RORgt, Treg differentiation by Foxp3, and Tfh differentiation by BCL6. Therefore, based on existing literature, high levels of BCL6 expression in mature T cells would be expected to lead to a Tfh-like phenotype. However, this type of differentiation was not observed in immature T cells, which was unexpected.

[0326] The cells were further engineered to express an anti-CD19 CAR to generate a series of "anti-CD19 infinite CAR T cells" (CD19 inCART). CD3- and CD8-infinite T cells, In1-L4a and Ie1-L4a, were engineered to express a chimeric antigen receptor (CAR) targeting human CD19 on their surface using a vector designated pJ3 plasmid (Figure 2A), resulting in the In1-L4aJ3 and Ie1-L4aJ3 infinite T cell lines. Both In1-L4aJ3 and Ie1-L4aJ3 T cells expressed the anti-CD19 CAR and were able to bind recombinant human CD19 protein (Figures 2B and 2C). The In1-L4aJ3 and Ie1-L4aJ3 infinite T cells were successfully generated, and they expanded in vitro with growth rates similar to their parental cells. Ie1-L4aJ3 demonstrated the ability to lyse the CD19-positive Raji lymphoma and Nalm6 leukemia cell lines in the presence of IL-2 at effector:target ratios of 0.2:1 and 1:1 (Fig. 3).

[0327] Example 2 - Modification of an In1-L4a derived T cell line to generate CD19 in CART cells In the following example, the modification of In1-L4a-derived infinite T cell line is described to generate CD19 in CAR T cells.These procedures can be used for other infinite T cells as well.However, for the sake of brevity, these procedures are only described in detail for In1-L4a and Ie1-L4a cell lines.Those skilled in the art can adapt the above method to insert anti-CD19 CAR gene into other infinite cell lines, or to insert other CARs or TCRs that target various tumor markers for various different tumors for therapeutic purposes.

[0328] A recombinant lentiviral vector expressing an anti-CD19 CAR driven by the MSCV promoter and hEGFRt was generated by the Gibson assembly method (NEB). The vector was named pJ3 (LV-MSCV-optimized C19-CD28z-T2A-tEGFR) (Figure 2A). To generate infectious pJ3 virus, 293T cells were co-transfected with the pJ3 plasmid and lentiviral vector packaging mix (ABM). One million In1-L4a and Ie1-L4a cells, as described in Example 1, were transduced with the pJ3 lentiviral vector. Ten days after transduction, CAR-positive cells were tested by flow cytometry using AF647-labeled anti-EGFR antibody (R&D) and FITC-labeled recombinant human CD19 protein (ACROBiosystems). The percentages of CAR-positive cells in the pJ3-transduced Ie1-L4a and In1-L4a groups were approximately 20% and 46.5% ( Figure 2B ).

[0329] The above CAR-positive percentages were further confirmed by double staining with FITC-labeled recombinant human CD19 protein and AF647-labeled cetuximab (Figure 2C). The CAR-positive cells were enriched by cell sorting using a cell sorter (BD). After sorting, relatively pure anti-CD19 CAR cells were collected and expanded in vitro (Figure 2D). In1-L4a and Ie1-L4a cells expressing CARs against human CD19 were designated In1-L4aJ3 and Ie1-L4aJ3, respectively. They showed exponential growth rates similar to those of their parental In1-L4a and Ie1-L4a indefinite T cells (Figure 1B).

[0330] In vitro cytotoxicity of CD19 in CAR T cells against CD19-positive lymphoma and leukemia cells: Raji cells are a CD19+ B-cell lymphoma cell line derived from a Burkitt lymphoma patient, and Nalm6 is a CD19+ B-cell leukemia cell line derived from an acute lymphoblastic leukemia patient, both of which are widely used in preclinical lymphoma research. Therefore, we used both of them to test the cytotoxic activity of the infinite anti-CD19 CART cell line by co-culturing effector and target cells at a ratio of 0.2:1 and 1:1 in the presence of IL-2. This test was performed in a 12-well plate. Briefly, 100,000 Raji or Nalm6 cells were cultured with 20,000 or 100,000 Ie1-L4aJ3 cells (anti-CD19 CART) or Ie1-L4a cells (without anti-CD19 CAR) per well in 2 mL of the above-mentioned medium. After 5 days of coculture, cells in each well were stained with APC-conjugated anti-CD8 antibody (BD) and acquired using a BD Fotessa Analyser (BD) to measure the percentage of viable T cells and tumor cells. Flow cytometry data were analyzed using FlowJo software. The data demonstrated that both Ie1-L4aJ3-infinite T cells could efficiently lyse both Raji and Nalm6 tumor cells in vitro (Figure 3). In contrast, no significant lysis of Raji or Nalm6 tumor cells was observed when Ie1-L4a cells were used, as they lack the anti-CD19 CAR.

[0331] Example 3 - Infinite T Cells for Universal Adoptive T Cell Therapy Infinite T cells have the capacity to proliferate rapidly for a long period of time.To date, we have generated infinite T cells from eight healthy donors by lentiviral transduction of BCL6 and BCL2L1 and observed that they could grow continuously and rapidly for >12 months in the presence of IL-2 or IL-15. Lentiviral incorporation of an anti-CD19 CAR into these cells did not affect the growth rate. The fold increase of these T cells was approximately 100-fold in 10 days and approximately 1 million-fold in 30 days, and their proliferation capacity remained unchanged over 12 months of continuous in vitro culture (Figure 5A). Phenotypically, infinite T cells are CD4 + T cells and CD8 + The CD4 T cells were mixed together and could be sorted to high purity using magnetic beads (Fig. 5B). + Foxp3 in T cells + The percentage of T cells that died was <5% (data not shown). Withdrawal of cytokines at any time point resulted in rapid cell death within 1 week, suggesting that these T cells had not transformed into a malignant phenotype or acquired the capacity for autonomous proliferation (Fig. 5C).

[0332] Infinite T cells exhibit high telomerase activity. Because T cell proliferation after 30–40 population doublings leads to gradual telomere shortening and replicative senescence (Barsov et al., 2011), we measured telomerase activity in these cells using the TRAPeze Telomerase Activity Detection Kit (Sigma). hTERT activity in infinite T cells was significantly higher than that in corresponding T cells derived from peripheral blood mononuclear cells (PBMCs) (Figure 6A). RNA-seq analysis of these cells was consistent with this observation in infinite CD4+ T cells, infinite CD8+ T cells, and infinite CD8+CAR+ T cells (Figure 6B). These results suggest that the transduced genes likely induce high telomerase activity in infinite T cells, thereby stabilizing telomere length, preventing replicative senescence, and conferring the characteristic of long-term proliferation.

[0333] Incorporation of an anti-CD19 CAR redirects the specificity of infinite T cells against B-cell malignancies.Lentiviral transduction of Infinite T cells with an anti-CD19 CAR (based on the clone FMC63 anti-CD19 scFv harboring the CD8α hinge / transmembrane domain, CD3ζ and CD28 signaling domains, and tEGFR as a transduction marker and safety switch (Wang et al., 2011)) enabled these cells to efficiently and specifically degranulate and kill the Daudi Burkitt lymphoma and NALM-6 acute B-cell lymphoblastic leukemia cell lines (Figures 7A-7B). Infinite T cells without the CAR did not exhibit significant cytotoxicity or degranulation. Compared with conventional CAR T cells generated from freshly isolated T cells from healthy donors, Infinite T cells were slower to kill tumor cells, but almost completely eliminated them by day 7 (Figure 7A). This delayed killing may be a promising clinical advantage, as it may result in fewer toxicities, such as cytokine release syndrome and neurotoxicity. These anti-CD19 infinite CAR T cells possessed a central memory and effector memory phenotype (Figure 7C) and showed very low or no expression of markers associated with T cell exhaustion (Figure 7D).

[0334] Transcriptional profile of infinite T cells. Corresponding CD4 isolated from PBMC samples + or CD8 + Infinite CD4 with or without anti-CD19 CAR compared to T cells + and / or CD8 + RNA-seq analysis of T cells was consistent with the flow cytometry and functional data, showing that they possess memory and cytotoxic phenotypes and do not express markers associated with classical T cell exhaustion (Figures 8A-8B). They differentiate from naive T cells to follicular helper T cells (T FH ) by overexpressing BCL6, the master transcription factor for differentiation into rhesus monkeys. 3 , these cells are T FH did not show the signature (Fig. 8A), and T FHInfinite T cells do not express high levels of CXCR5 (Figure 8C), a characteristic of infinite T cells (Nurieva et al., 2009; Rawal et al., 2013). However, they retain expression of the chemokine receptors CCR4 and CCR7, which are important for T cell trafficking to lymph nodes, and CXCR4, which is important for trafficking to the bone marrow (Figure 8C) (Viola et al., 2006). Both sites are commonly involved in lymphoma. Infinite T cells do not express senescence markers such as B3GAT1 (CD57), CD160, or KLRG1 (Figure 8D) (Xu et al., 2017). The expression profiles of chemokine (Figure 9A) and cytokine (Figure 9B) genes were largely similar between infinite T cells and corresponding CD4 or CD8 T cells derived from peripheral blood. Cytokine receptor gene expression showed some differences in infinite T cells compared with corresponding CD4 or CD8 T cells from peripheral blood, including, but not limited to, increased levels of IL2RA, IL15RA, and IL21R, and decreased levels of IL4R, IL7R, IL10RA, IL17RA, IL18R1, and IFNGR1 (Figure 9C).

[0335] Infinite CAR T cells retain proliferative and cytotoxic function after freeze-thaw. Infinite T cells with and without CAR were cryopreserved and thawed after 6 months. After thawing, they showed strong CAR expression when anti-EGFR antibodies were used (Figure 10A). When these cells were cultured in IL-2, the cell number increased approximately 100-fold in 10 days, confirming that the proliferative capacity of infinite CD8 CAR T cells was maintained after freeze-thawing (Figure 10B). Furthermore, these cells were shown to exhibit highly significant and specific cytotoxic activity against malignant B cells (Figure 10C).

[0336] Infinite γδ T cells do not express exhaustion markers. Unlimited γδ T cells did not significantly express classical T cell exhaustion markers (Figure 11).

[0337] Anti-CD19 infinite CAR T cells demonstrate anti-tumor efficacy in in vivo models.Using luciferase-labeled infinite CAR T cells, we observed that after intraperitoneal (ip) injection into NSG mice, these T cells rapidly disappeared within 72 hours without cytokine support as monitored by bioluminescence imaging (BLI) (Figure 12, center column). This is likely because mouse cytokines (both IL-2 and IL-15) do not support human T cell growth. In contrast, injection of recombinant human IL-15 on days 1 and 3 induced robust T cell proliferation, with cells persisting for a week after IL-15 withdrawal (Figure 12, right column). These results suggest that IL-15 promotes proliferation and persistence in vivo and that low doses may be sufficient. Similar effects were observed with IL-2.

[0338] Next, we transfected 3 × 10 luciferase-labeled NALM-6 tumor cells with or without CAR. 6 NSG mice were intravenously (IV) injected with infinite T cells / mouse and IL-15 on days 0, 4, 7, and 11. Mice treated with infinite CAR T cells showed significant tumor control and prolonged survival compared to infinite T cells without CAR (Figure 13). Collectively, these results provide a rationale for engineering infinite T cells to secrete IL-2 or IL-15 to enhance in vivo expansion and persistence.

[0339] Microbial-associated and tumor-associated antigen-specific infinite T cells.Tetramer testing of infinite T cells generated from HLA-A2+ donors revealed the presence of a mixture of microbial- and tumor-associated antigen-specific T cells (Figure 14). To generate an enriched population of these T cells, we stimulated healthy donor peripheral blood mononuclear cells from HLA-A2+ donors with a pool of peptides derived from EBV proteins. After 24 hours, CD137-positive T cells were sorted and used to generate infinite T cells by transducing them with the lentiviral vector L5x (Figure 22), which expresses BCL6 and BCL2L1. The virus production and transduction protocols were described in Example 1. Two weeks after transduction, the transduced T cells were restimulated with a CD3 / CD28 / CD2 T cell activator and then continued to be cultured as described in Example 1. After 7 weeks of in vitro culture and expansion in the presence of IL-2, the expanded cells were stained with three APC-labeled tetramers (including the BMLF1-HLA-A2 tetramer) and enriched with APC-enriched magnetic beads. The enriched infinite T cells were continuously cultured like all other infinite T cells. At week 13, the enriched infinite T cells were stained with the APC-labeled BMLF1-HLA-A2 tetramer and found to be CD8 positive and BMLF1-HLA-A2 tetramer positive, suggesting that they were specific for the HLA-A2-binding peptide (GLCTLVAML) derived from the EBV-BMLF1 protein (Figure 15). Similar approaches can be used to generate other antigen-specific T cells against microbial- and tumor-associated antigens. These antigen-specific T cells can then be transduced with the desired CAR or TCR to generate dual antigen-specific T cells.

[0340] Tet-off system as a safety switch.We did not observe malignant transformation or cytokine-independent proliferation of infinite T cells in vitro, even after 6 to >12 months of culture of infinite T cells derived from eight donors (Figure 4). However, to ensure safety for clinical translation, we incorporated a Tet-off safety switch that could turn off the transduced BCL6 and BCL2L1 genes using doxycycline. Even after incorporating this Tet-off safety switch, infinite T cells maintained their growth rate in the absence of doxycycline but stopped proliferating and gradually underwent cell death in the presence of 1 μg / mL doxycycline (Figure 16). This doxycycline concentration is achievable with standard therapeutic doses of doxycycline in humans (Agwuh et al., 2006). Light microscopy imaging revealed that the infinite T cells gradually decreased in size, along with a decrease in proliferative clusters, with increasing doxycycline concentrations (Figure 17). Furthermore, CD25 expression was significantly decreased in the presence of doxycycline (Figure 17), and PD-1 expression was increased, suggesting that the BCL6 and / or BCL2L1 genes likely regulated the expression of these molecules. Expression of other T cell co-inhibitory receptors was not significantly altered in the presence of doxycycline (Figure 18). Similar tet-off safety switches can also be used to regulate the IL-2 or IL-15 cytokine genes integrated into the indeterminate T cells.

[0341] Anti-CD19 infinite CAR T cells produce effector cytokines in response to B-cell tumor cells. To characterize the cytokine profile of infinite T cells generated in response to tumor cells, we investigated the effector:target ratio of 5:1 and expressed CD8 T cells transduced with or without an anti-CD19 CAR. +Infinite T cells were co-cultured with NALM-6 tumor cells. After 3 days, cytokine levels in the supernatant were measured. Results showed that most Infinite T cells bearing an anti-CD19 CAR produced significant amounts of IL-2, GM-CSF, IFN-γ, IL-5, and IL-17 in response to NALM-6 tumor cells, whereas Infinite T cells without an anti-CD19 CAR did not (Figure 19). Production of TNF-α, IL-4, IL-6, IL-10, or IL-13 by anti-CD19 Infinite CAR T cells in response to tumor cells was minimal or not significantly different from Infinite T cells without CAR expression. However, Infinite T cells with or without CAR expression produced large amounts of IL-4 (>10,000 pg / mL) in the presence or absence of tumor cells (Figure 19 and data not shown). Because IL-4 can suppress inflammation induced by T cells, macrophages, and other immune cells, this property of infinite T cells to constitutively produce large amounts of IL-4 in the absence of external stimuli may potentially have clinical applications for treating various inflammatory disorders (e.g., autoimmune diseases, graft-versus-host disease, certain types of infections associated with cytokine release syndrome, toxicity associated with CAR T cell therapy and other adoptive T cell therapies, inflammatory bowel disorders, immune-related adverse events associated with various immunotherapies, hemophagocytic lymphohistiocytosis, periodic fever syndromes, etc.).

[0342] tEFGR safety switch for anti-CD19 infinite CAR T cells. To determine whether cleaved EGFR (tEGFR) can act as a safety switch for infinite T cells, we co-cultured infinite T cells expressing anti-CD19 CAR and tEGFR with or without natural killer (NK) cells isolated from peripheral blood mononuclear cells of healthy donors in the presence of cetuximab at a concentration of 5 μg / mL. Cetuximab induced significant lysis of anti-CD19 infinite CAR T cells by antibody-dependent cellular cytotoxicity (ADCC) compared with rituximab used as a control (Figure 20). These results suggest that tEGFR can act as a safety switch to eliminate infinite T cells in vivo in the event of an adverse event.

[0343] Generation of infinite T cells by transduction of BCL6 and BIRC5 genes. We observed that infinite T cells could be generated by transducing human T cells with the BCL6 and BCL2L1 genes or the BCL6 and BIRC5 genes (Figure 21A). BCL2L1 encodes the anti-apoptotic protein Bcl-xL, and BIRC5 encodes survivin, an inhibitor of apoptosis (IAP) family protein that promotes proliferation and prevents cell apoptosis. Transduction of either gene combination generated infinite T cells with comparable long-term proliferation capacity at exponential growth rates in the presence of IL-2 (Figure 21B). Furthermore, these infinite T cells were generated using a Tet-off safety switch that could turn off the transduced BCL6 and BCL2L1 or BCL6 and BIRC5 genes by using doxycycline. The vector also contained the IL-15 gene, which was transduced into these cells. Th cells grew at an exponential rate in the absence of doxycycline, but in the presence of 1 μg / mL doxycycline, they stopped proliferating and gradually underwent cell death, despite transduction with IL-15 and the addition of IL-2 to the culture medium (Figure 21C).

[0344] An example of a construct containing BCL6 and Bcl-xL, L5x (MSCV-BCL6-P2A-BCL-xL-T2A-rtTA), contains at least a P2A element between wild-type BCL-6 and BCL-xL, and a T2A element between BCL-xL and rtTA (Tet on transactivator) (Figure 22).

[0345] Figure 23 provides several examples of construct embodiments including at least BCL6; such examples may or may not use BCL-xL. By way of example only, in Example 1, an MSCV promoter is used to control the overexpression of BCL6 and rtTA, and an H1 promoter controls a caspase 9-targeting shRNA to knock down caspase 9 expression. In Example 2, an MSCV promoter is used to control the overexpression of BCL6 and rtTA, in addition to a human U6 promoter to control a BAK gene-targeting shRNA to knock down BAK expression. In Example 3, an MSCV promoter controls the overexpression of BCL6, HSP27, and rtTA. In Example 4, an MSCV promoter controls the expression of BCL6 and rtTA, and a U6 promoter controls the expression of miRNA21.

[0346] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More particularly, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Agwuh KN, MacGowan A. Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J Antimicrob Chemother. 2006;58(2):256-265. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Barsov EV. Telomerase and primary T cells: biology and immortalization for adoptive immunotherapy. Immunotherapy. 2011;3(3):407-421. Hooijberg, E., et al., J Immunol, 165(8): p. 4239-45, 2000. Hurton, L.V., et al., Proc Natl Acad Sci U S A, 113(48): p. E7788-e7797, 2016. Migliaccio, M., et al., J Immunol, 165(9): p. 4978-84, 2000. Nurieva RI, Chung Y, Martinez GJ, et al. Bcl6 mediates the development of T follicular helper cells. Science. 2009;325(5943):1001-1005. Rawal S, Chu F, Zhang M, et al. Cross talk between follicular Th cells and tumor cells in human follicular lymphoma promotes immune evasion in the tumor microenvironment. J Immunol. 2013;190(12):6681-6693. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001. Viola A, Contento RL, Molon B. T cells and their partners: The chemokine dating agency. Trends Immunol. 2006;27(9):421-427. Wang X, Chang WC, Wong CW, et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood. 2011;118(5):1255-1263. Xu W, Larbi A. Markers of T Cell Senescence in Humans. Int J Mol Sci. 2017;18(8). WO2000 / 14257 WO2012 / 129514 WO2013 / 126726 WO2013 / 071154 WO2013 / 123061 WO2013 / 166321 WO2014 / 031687 WO2014 / 055668 U.S. Patent Application Publication No. 2002131960 U.S. Patent Application Publication No. 2013287748 U.S. Patent Application Publication No. 20130149337 U.S. Patent No. 6,410,319 U.S. Patent No. 6,451,995 U.S. Patent No. 7,070,995 U.S. Patent No. 7,265,209 U.S. Patent No. 7,354,762 U.S. Patent No. 7,446,179 U.S. Patent No. 7,446,190 U.S. Patent No. 7,446,191 U.S. Patent No. 8,252,592 U.S. Patent No. 8,324,353 U.S. Patent No. 8,339,645 U.S. Patent No. 8,398,282 U.S. Patent No. 8,479,118 European Patent Application No. 2537416

Claims

1. B-cell lymphoma 6 (BCL6) and (a) B-cell lymphoma 2 (BCL-2) family genes; or (b) survivin, A composition comprising T cells engineered to express

2. 2. The composition of claim 1, wherein the BCL-2 family gene is BCL2L1 (Bcl-xL), BCL-2, MCL1, BCL2L2 (Bcl-w), BCL2A1 (Bfl-1), BCL2L10 (BCL-B), or a combination thereof.

3. the T cells produce IL-4 in the absence of external stimuli, and / or the T cells are engineered to express one or more cytokines; The composition according to claim 1 or 2.

4. (a) the T cells are derived from a donor who has not been diagnosed with cancer; and / or (b) the T cells are derived from an individual in need of treatment; The composition according to any one of claims 1 to 3.

5. (a) the T cells are CD4+ T cells, CD8+ T cells, iNKT cells, NKT cells, γδT cells, regulatory T cells, innate lymphoid cells, or combinations thereof; and / or (b) the T cells comprise CD4+ cells, CD8+ cells and / or γδ T cells; and / or (c) the T cells are naive T cells, effector T cells, memory T cells, stem cell memory T cells, terminally differentiated T cells, or a combination thereof; and / or (d) the T cells are TCRαβ cells, TCRγδ T cells, or a combination thereof; and / or (e) the T cells are Th1 / Tc2, Th2 / Tc2, Th9 / Tc9, Th17 / Tc17, Tfh, Th22, Tc22, or a combination thereof; and / or (f) the T cells express cytokines and cytotoxic molecules that are IFNγ, GM-CSF, TNFα, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-16, IL-17, IL-23, IL-32, granzyme B, perforin, or a combination thereof; (g) the T cells are specific for one or more microbial antigens, one or more autoantigens, or one or more tumor antigens; and / or (h) the T cells express one or more genetically engineered antigen receptors; The composition according to any one of claims 1 to 4.

6. 6. The composition of any one of claims 1 to 5, wherein the T cells are engineered to express one or more chimeric antigen receptors (CARs) and / or one or more T cell receptors (TCRs).

7. (a) the CAR is selected from the group consisting of CD8a, CD28, PD-1, CTLA4, the alpha, beta, or zeta chain of the T cell receptor, CD2, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8b, CD9, CD16, CD22, CD27, CD32, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD1 and / or a partial or complete sequence of any of the following: 54, CD160, BTLA, LAIR1, TIGIT, TIM4, ICOS / CD278, GITR / CD357, NKG2D, LAG-3, PD-L1, PD-1, TIM-3, HVEM, LIGHT, DR3, CD30, CD224, CD244, SLAM, CD226, hinge of DAP, or combinations or synthetic molecules thereof; (b) the CAR comprises a partial or complete transmembrane domain of the T cell receptor alpha chain, the T cell receptor beta chain, the T cell receptor zeta chain, CD28, CD2, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, PD-1, CTLA4, DAP, a synthetic molecule, or a combination thereof; and / or (c) the CAR comprises one or more costimulatory domains of CD28, CD27, OX-40 (CD134), DAP10, DAP12, 4-1BB, or a combination thereof. The composition of claim 6.

8. (a) the composition comprises between 100,000 and 10 billion T cells; and / or (b) the T cells comprise one or more safety switches; and / or (c) the T cells express IL-2, IL-15, one or more growth factors, one or more differentiation factors, or a combination thereof; and / or (d) the cells maintain a proliferation rate for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more; and / or (e) the T cells have enhanced anti-tumor cytotoxicity, cytokine production, in vivo proliferation, in vivo persistence, and / or improved function. The composition according to any one of claims 1 to 7.

9. 9. An in vitro method for producing the T cells of any one of claims 1 to 8, comprising the step of introducing into the cells one or more vectors encoding BCL6 and (a) a BCL-2 family gene or (b) survivin, wherein the BCL-2 family gene is BCL2L1 (Bcl-xL), BCL-2, MCL1, BCL2L2 (Bcl-w), BCL2A1 (Bfl-1), BCL2L10 (BCL-B), or a combination thereof.

10. (a) the vector links BCL6 and Bcl-xL with a 2A sequence, and / or (b) the vector is a lentiviral vector, and / or (c) the introducing step comprises transducing the cells with the lentiviral vector in the presence of IL-2, IL-15 and / or one or more other growth factors; and / or (d) the method further comprises activating the T cells with CD3 and CD28; and / or (e) the method further comprises culturing the cells in the presence of IL-2 and / or IL-15; and / or (f) the cells are cultured for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more without essentially a decrease in proliferation rate; and / or (g) the method further comprises sorting for T cell subsets; and / or (h) the method further comprises the step of introducing one or more cytokines and / or one or more safety switches into the T cells, wherein (i) the one or more cytokines and / or one or more safety switches are present on the same vector as the BCL6 and (a) a BCL-2 family gene or (b) survivin, or (ii) the one or more cytokines and / or one or more safety switches are present on a vector different from the BCL6 and (a) a BCL-2 family gene or (b) survivin.

10. The method of claim 9.

11. 9. A composition comprising the population of cells of any one of claims 1 to 8 for treating an immune-related disorder, an infectious disease and / or cancer, wherein the immune cells are targeted against one or more molecules.

12. 10. A composition comprising the T cells of any one of claims 1 to 8 for use in a method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a composition comprising the T cells.

13. (a) the disease or disorder is an infectious disease, cancer, or immune-related disorder, wherein (i) the immune-related disorder is an autoimmune disorder, graft-versus-host disease, allograft rejection, or an inflammatory condition, or (ii) the disease is cancer, wherein the cancer is a solid cancer or a hematological malignancy, or (b) the disease or disorder is an autoimmune disease, graft-versus-host disease, infections associated with cytokine release syndrome, immunotherapy-associated toxicity, inflammatory bowel disorder, immune-related adverse events associated with immunotherapy, hemophagocytic lymphohistiocytosis, periodic fever syndrome, or a combination thereof; 13. A composition for use according to claim 12.

14. (a) the T cells are allogeneic immune cells to the subject; or (b) the T cells are autologous immune cells to the subject; 14. A composition for use according to claim 12 or 13.

15. (a) the T cells produce IL-4 under conditions in which inflammation induced by T cells, macrophages and / or other immune cells should be suppressed; and / or (b) the method further comprises administering to the subject at least a second therapeutic agent; A composition for use according to any one of claims 12 to 14.

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