Immune cells for adoptive cell therapy

Abstract

Methods are provided for generating unlimited immune cells with increased lifespan and high proliferation rate by modifying them to express BCL6 and genes that promote cell survival. Further methods are provided for generating and using said unlimited immune cells to treat diseases such as cancer.

Description

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

[0002] 1. Field of Invention

[0003] In general, this disclosure covers at least the fields of molecular biology, cell biology, immunology, and medicine. More specifically, it relates to methods for generating an unlimited number of immune cells and their use.

[0004] 2. Relevant Technical Descriptions

[0005] NK cells and T cells are two types of cytotoxic lymphocytes frequently used in adoptive cell therapy research. CAR-NK cells, CAR T cells, TCR-transduced T cells, and T cells with endogenous T-cell receptors derived from NK cells and T cells, specific to microorganisms or tumor antigens, are promising approaches for treating both hematologic malignancies and solid tumors. Three CD19-targeting CAR-T cell products have recently been approved by the FDA for B-cell malignancies, and more products are under development. Currently, the generation of TCR-T cells and CAR-T cell therapy products is a multi-step process that requires first isolating T cells from healthy donors or patients, then introducing TCRs or CARs into those T cells using viral or non-viral vectors, and expanding the genetically modified T cells in vitro before infusion into the patient. Similarly, the generation of microbial and tumor antigen-specific T cells is also a multi-step process that requires first collecting T cells from a healthy donor or patient, then isolating and / or stimulating them in vitro with microbial or tumor antigenic peptides or proteins, and then expanding the T cells in vitro before infusion into the patient.

[0006] This makes preparing products for each patient expensive, cumbersome, and time-consuming. Furthermore, the T cells produced in this way can only expand in vitro for a few weeks before they become senescent, thus limiting the number of microbe and tumor antigen-specific T cells, TCR-T cells, or CAR-T cells that can be produced from each patient or healthy donor.

[0007] Recent reports suggest that factors that promote CAR-T cell survival through genetic modification are positively correlated with better therapeutic efficacy (Hurton et al., 2016). Therefore, strategies to increase the lifespan of normal and / or genetically modified T cells while maintaining their proliferation, cytokine production, and cytotoxic functions could significantly reduce the time required to generate them and the cost of adoptive T-cell therapy approaches, while potentially increasing their potency. While the cytotoxic T-cell line, TALL-104 (US Patent No. US5272082), and the NK-cell line, NK-92 (US Patent Publication No. US20020068044), can proliferate indefinitely and possess cytotoxic activity, they are derived from T-cell and NK-cell leukemia, respectively. Therefore, these cell lines contain mutations and other genetic alterations and are unsafe for therapeutic use in humans. Thus, there is an unmet need for strategies to achieve these goals by increasing the lifespan of normal T cells. Invention Overview

[0008] In one embodiment, this disclosure provides a composition comprising immune cells, including at least T cells or NK cells, modified to have an increased lifespan compared to unmodified immune cells. Such cells may be referred to herein as unlimited cells. In particular embodiments, the methods and compositions relate to immune cells having the expression of B-cell lymphoma 6 (BCL6) and pro-survival genes or anti-apoptotic genes or genes promoting cell survival, including heterologous expression. As used herein, a pro-survival gene refers to a nucleic acid polymer capable of exerting an anti-apoptotic function or promoting survival through any mechanism. Nucleic acid polymers capable of exerting an anti-apoptotic function may be one or more of the Bcl2 family genes, such as BCL-xL (also known as the BCL2L1 gene), BCL-2, MCL1, BCL2L2 (Bcl-w), BCL2A1 (Bfl-1), BCL2L10 (BCL-B), etc. Nucleic acid polymers capable of anti-apoptotic function can be one or more genes from the inhibitor of apoptosis (IAP) family, such as XIAP, BIRC2 (C-IAP1), BIRC3 (C-IAP2), NAIP, and BIRC5 (survival protein). These anti-apoptotic nucleic acid polymers can 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 polymers used for knockdown or knockout can be shRNA expression cassettes, or these caspase genes can be knocked out using gene editing methods (CRISPR, TALEN, zinc finger methods, etc.). Nucleic acid polymers that can exert anti-apoptotic functions can 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 that can exert anti-apoptotic functions can have anti-apoptotic effects, such as 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.

[0009] In a particular embodiment, the cells covered herein are capable of constitutively producing large amounts of IL-4 (e.g., greater than 1000 pg / mL in in vitro cultures when incubated at a cell concentration of 10,000 cells / mL) in the absence of external stimuli, and such cells can be used for clinical applications, such as for the treatment of various inflammatory conditions, including autoimmune diseases, graft-versus-host disease, certain types of infections associated with cytokine release syndrome, toxicity associated with CAR T-cell and other adoptive T-cell therapies, inflammatory bowel disease, immune-related adverse events associated with various immunotherapies, hemophagocytic lymphohistiocytosis, periodic fever syndrome, etc., because IL-4 can suppress inflammation induced by T cells, macrophages, and other immune cells.

[0010] In some aspects, the gene promoting cell survival is an anti-apoptotic B-cell lymphoma 2 (BCL-2) family gene. In some aspects, 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 a particular aspect, the anti-apoptotic BCL-2 family gene is Bcl-xL.

[0011] In a further aspect, the T cells or NK cells are further modified to express IL-2 and / or IL-15.

[0012] In some respects, the T cells or NK cells are derived from a healthy donor (e.g., a donor not diagnosed with cancer). In other respects, the T cells or NK cells are derived from a patient. In a particular respect, the donor is a human being.

[0013] In a particular aspect, the T cells include CD4+ T cells, CD8+ T cells, iNKT cells, NKT cells, γδT cells, regulatory T cells, innate lymphoid cells, or combinations thereof. In some aspects, the T cells include CD8 and / or γδT cells. The T cells are naive T cells, effector T cells, memory T cells, stem cell memory T cells, terminally differentiated T cells, or combinations thereof. In some aspects, the T cells are TCRαβ cells or TCRγδT cells. In some aspects, the composition has no or substantially no follicular helper (Tfh) T cells. In some aspects, the immune cells consist of T cells that are Th1 / Tc1, Th2 / Tc2, Th9 / Tc9, Th17 / Tc17, Tfh, Th22, Tc22, or combinations thereof. In a particular aspect, the T cells express IFNγ, granzyme B, perforin, or combinations thereof.

[0014] In some respects, the T cells or NK cells are virus-specific or tumor antigen-specific. In some respects, the T cells or NK cells are further modified to express one or more CARs and / or one or more TCRs. In some respects, the CAR or TCR comprises 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, FRα, LeY, HER2, IL13Rα2, DLL3, DR5, FAP, LMP1, MAGE-A1, MAGE-A4, MG7, MUC16, PMEL, ROR2, VEGFR2, AFP, EphA2, PSCA, EPCAM, EGFR, PSMA, EGFRvIII, GPC3, CEA, GD2, NY-ESO-1, TCL1, mesothelin, or BAFF-R antigen-binding regions. In a particular aspect, the CAR includes a CD19 antigen-binding region.

[0015] In some respects, 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 mixtures thereof.

[0016] In another aspect, the immune cells contain at least one safety switch. In some aspects, the safety switch is a truncated EGFR (e.g., an EGFR lacking domains 1 and 2). In some aspects, the immune cells (T cells, innate lymphoid cells, and / or NK cells) express IL-2, IL-15, other growth or differentiation factors, or combinations thereof.

[0017] In some respects, the cells maintain a proliferation rate of 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 thereof. In some respects, the immune cells possess enhanced antitumor cytotoxicity, in vivo proliferation, in vivo persistence, and / or improved function.

[0018] In another embodiment, a method for generating T cells, innate lymphoid cells, or NK cells according to embodiments of the present invention is provided, comprising introducing a vector encoding BCL6 and a gene promoting cell survival into the cells. In some aspects, the gene promoting cell survival is an anti-apoptotic B-cell lymphoma 2 (BCL-2) family gene. In some aspects, 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 particular aspect, the anti-apoptotic BCL-2 family gene is Bcl-xL. In some aspects, the vector links BCL6 and Bcl-xL to a 2A sequence. In a particular aspect, the 2A sequence is a T2A sequence.

[0019] In some aspects, the vector is a lentiviral vector. In some aspects, introduction includes transducing the cells with a lentiviral vector in the presence of IL-2 and / or other growth factors. In some aspects, IL-2 is present at concentrations from 10 IU / mL to 1000 IU / mL, for example, 10-50 IU / mL, 50-75 IU / mL, 75-100 IU / mL, 100-250 IU / mL, 250-500 IU / mL, 500-750 IU / mL, or 750-1000 IU / mL. In particular aspects, IL-2 is present at concentrations of 100, 200, 300, 400, or 500 IU / mL.

[0020] In another aspect, the method further includes activating the T cells with CD3 and CD28. In some aspects, the method further includes culturing the cells in the presence of IL-2 and / or IL-15. In some aspects, the IL-2 and / or IL-15 are present at concentrations 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 aspects, the cells are cultured for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (or any range therebetween) without a substantial decrease in proliferation rate.

[0021] In a further aspect, the method further includes sorting T cell subclasses. In a particular aspect, the T cell subclasses include CD4+ T cells, CD8+ T cells, and / or γδ T cells.

[0022] The embodiments include compositions comprising cell populations of the embodiments of the present invention (e.g., immune cells modified to express B-cell lymphoma 6 (BCL6) and genes that promote cell survival) for the treatment of immune-related conditions, infectious diseases and / or cancer.

[0023] The implementation scheme relates to a method of treating a disease or condition in a subject, which includes administering to the subject an effective amount of immune cells of the present invention (e.g., immune cells modified to express B-cell lymphoma 6 (BCL6) and genes that promote cell survival).

[0024] In some aspects, the disease or condition is an infectious disease, cancer, and / or an immune-related condition. In some aspects, the immune-related condition is an autoimmune disease, graft-versus-host disease, allogeneic graft rejection, or other inflammatory conditions. In some aspects, the immune cells are allogeneic. In particular, the immune-related condition is cancer. For example, the cancer is a solid tumor or a hematologic malignancy.

[0025] In another aspect, the method further includes administering at least a second therapeutic agent. In some aspects, the at least second therapeutic agent includes chemotherapy, immunotherapy, surgery, radiotherapy, drug therapy, hormone therapy, biological therapy, or combinations thereof.

[0026] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although preferred embodiments of the invention are indicated) are given by way of example only, as 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 description of the attached diagram

[0027] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. The invention can be better understood by referring to one or more of these figures and the detailed description of specific embodiments presented herein in combination with them.

[0028] Figure 1A-1G :( Figure 1A A map of a lentiviral vector containing the BCL6-T2A-BCL-xL gene driven by the human PGK promoter. Figure 1B The diagram illustrates the proliferation rate of the unlimited T cell line. The top left panel shows the growth curves of In1-L4a (unlimited CD3 T cells) and Ie1-L4aJ3 (unlimited CD8 CAR-T) at month 2 in the presence of 400 IU / mL IL-2. The top right panel shows the growth curves of unlimited CD8 CAR T cells (Ie1-L4aJ3) in the presence of 100 ng / mL IL-15, IL-7, and IL-21, or in the absence of cytokines. The data show that unlimited T cells grow in the presence of IL-15, but do not grow in the presence of IL-7, IL-21, or in the absence of cytokines. The lower left and lower right plates show that unlimited T cells (including CD4 unlimited αβ T cells, CD8 unlimited αβ T cells (Ie1-L4a), CD8 unlimited αβ CAR-T cells (Ie1-L4aJ3), unlimited γδ T cells (Igd1-L4a), and unlimited γδ CAR-T cells (Igd1-L4aJ3)) continued to proliferate in vitro in the presence of IL-2 at month 5. Figure 1C The diagram illustrates the phenotype of the unlimited T cell line In1-L4a, as determined by the expression of CD3, CD4, CD8, CD16, CD56, TCRαβ, and TCRγδ. Figure 1D The diagram illustrates the phenotype of sorted γδ T cells obtained using anti-TCRγδ antibody. It shows the expression of TCRγδ, TCRαβ, and CD16 in these cells. Figure 1E The diagram illustrates the major subclasses of sorted γδT cells obtained using anti-TCRγ9 and anti-TCRδ2 antibodies. The majority of unlimited γδT cells are positive for TCRγ9δ2. Figure 1F The diagram illustrates the phenotype of unlimited T cells at month 4. Most of them are effector and central memory T cells, primarily expressing IFNγ, granzyme B, and perforin. Figure 1G The diagram illustrates the expression of various co-inhibitory receptors on unlimited CAR-T cells.

[0029] Figure 2A -2E:( Figure 2A A map of the lentiviral vector pJ3 containing anti-CD19 CAR and a truncated human EGFR expression cassette. Figure 2B The figure illustrates the CAR-positive percentage of Ie1-L4aJ3 (unlimited CD8 CART) and In1-L4aJ3 (unlimited CD3 CART) transduced via the lentiviral vector pJ3. The CAR-positive percentage was determined by flow cytometry 10 days post-transduction using either FITC-labeled human CD19 protein or anti-EGFR antibody. Figure 2C The diagram illustrates the percentage of CAR-positive cells for In1-L4aJ3 (unlimited CD3CAR). tEGFR was stained with cetuximab labeled with AF647, and anti-CD19 CAR was stained with recombinant human CD19 protein labeled with FITC. Figure 2D The diagram illustrates the percentage of In1-L4aJ3 (unlimited CD3CAR) CAR-positive cells before and after sorting. tEGFR was stained with AF647-cetuximab, and anti-CD19 CAR was stained with recombinant human CD19 protein labeled with FITC.

[0030] Figure 3 The diagram illustrates the in vitro cytotoxicity of Ie1-L4aJ3 (unlimited CD8 CART) against Raji and Nalm6 cells in 12-well plates at effector:target (E:T) ratios of 0.2:1 and 1:1. Ie1-L4aJ3 (unlimited CD8 CART) cells or control Ie1-L4a (unlimited CD8 T cells without CAR) cells were co-cultured with Raji or Nalm6 cells for 5 days. The percentage of tumor cells in the co-culture is shown on days 0, 1, 3, and 5.

[0031] Figure 4The diagram illustrates the in vitro cytotoxicity of unlimited T cells 4 months after expansion. Ie1-L4a (unlimited CD8 T cells), Ie1-L4aJ3 (unlimited CD8 CAR-T), Igd1-L4a (unlimited γ / δ T cells), or Igd1-L4aJ3 (unlimited γδ CAR-T cells, CAR-T percentage >90%) cells were co-cultured with Daudi or Nalm6 cells for 7 days in the presence of IL-15 at an effector:target (E:T) ratio. The percentage of tumor cells in the co-culture is shown on days 0, 1, 2, 4, and 7. These results suggest that: 1) CD8 unlimited CAR-T and γδ unlimited CAR-T cells retain specific cytotoxicity even after prolonged in vitro culture and expansion; and 2) γδ unlimited T cells without CAR but possessing endogenous γ9δ2 TCR or other TCRs can induce lysis of certain types of tumor cells, possibly mediated by γδ TCR. For example, Daudi cells can be killed by γδ unlimited T cells without CAR, while Nalm-6 can only be killed by γδ unlimited T cells transduced with CAR. Besides some lymphoma tumor cells, some myeloma cell lines and other cancer cell lines are also known to be killed by γδ T cells.

[0032] Figures 5A-5C (Fig. 5A) Growth rate of unlimited T cells (CD4+CD8 or CD8) with or without anti-CD19 CAR in the presence of IL-2. (Fig. 5B) Unlimited T cells are a mixture of CD4 and CD8 T cells (left plate) and can be sorted to high purity, as shown for CD8 unlimited T cells (right plate). (Fig. 5C) Unlimited T cells cultured for 6 months were then incubated without IL-2 (shown) or IL-15 (not shown). The cell number decreased rapidly within 6 days, suggesting no evidence of autonomous growth or malignant transformation of unlimited T cells even after long-term in vitro culture.

[0033] Figures 6A-6B (Figure 6A) Telomerase activity was measured in unlimited T cells or peripheral blood mononuclear cells (PBMCs) using the TRAPEze Telomerase Activity Assay Kit, following the manufacturer's instructions. (Figure 6B) Genes associated with telomerase activity, as determined by RNAseq analysis in unlimited T cells or corresponding PBMC samples, are shown as a heatmap. These results suggest that unlimited T cells possess very high telomerase activity.

[0034] Figures 7A-7D: (Figure 7A) Unlimited T cells with or without anti-CD19 CAR, or CAR T cells generated from peripheral blood T cells using conventional methods, were labeled with CellTrace FarRed, and Daudi tumor cells were labeled with CellTrace Violet, and co-cultured at a 1:1 effector:target ratio. The percentage of viable tumor cells was determined at 3, 5, and 7 days (bottom right). The absolute number of viable tumor cells was also calculated by flow cytometry using CountBright Absolute counting beads (ThermoFisher Scientific), and the results are consistent with the percentage of viable tumor cells shown. (Figure 7B) Unlimited T cells with or without anti-CD19 CAR were co-cultured with NALM-6B cell leukemia cells at a 1:1 ratio. Degranulation was determined by CD107a staining after 6 hours. These results suggest that CAR-expressing unlimited T cells are highly cytotoxic and degranulate in response to B-cell tumors. (Figures 7C and 7D) The phenotype of anti-CD19 unlimited CAR T cells was determined by flow cytometry based on the displayed markers. Anti-CD19CAR expression was determined by staining with fluorescently labeled recombinant human CD19-Fc protein. The results showed that unlimited T cells did not express high levels of conventional exhaustion markers such as CTLA-4, PD-1, TIM-3, CD160, or 2B4 (CD244).

[0035] Figures 8A-8D: Genes or gene signatures associated with T cell subclasses (Figure 8A), exhaustion markers (Figure 8B), chemokine receptors (Figure 8C), and aging markers (Figure 8D) as heatmaps in unlimited T cells or corresponding PBMC samples, as determined by RNAseq analysis.

[0036] Figures 9A-9C Genes associated with chemokine expression (Fig. 9A), cytokine expression (Fig. 9B), and cytokine receptors (Fig. 9C) in unlimited T cells or corresponding PBMC samples, as determined by RNAseq analysis, are displayed as a heatmap.

[0037] Figures 10A-10C(Fig. 10A) Unlimited T cells or CAR-transduced T cells were thawed, and anti-CD19 CAR expression was determined by anti-EGFR antibody staining. (Fig. 10B) Growth rate of anti-CD19 unlimited CAR T cells after thawing and in vitro culture with IL-2. Cell number in culture at different days is shown. (Fig. 10C) Cytotoxic activity of cells thawed in A was determined after 4 days of 1:1 co-culture between unlimited T cells and NALM-6 tumor cells, as described below Fig. 7A. Gates show the percentage of viable tumor cells.

[0038] Figure 11 The phenotype of unlimited γδ T cells (bottom panel) was determined by flow cytometry based on the displayed markers and compared with the corresponding γδ T cells from healthy donor PBMCs (top panel). The results showed that unlimited γδ T cells did not express high levels of conventional exhaustion markers.

[0039] Figure 12 Unlimited T cells labeled with luciferase were injected intraperitoneally (ip) on days 1 and 3, with or without IL-15 injection. T cell numbers were imaged using bioluminescence imaging (BLI). Results showed that IL-15 promoted the growth and expansion of unlimited T cells in vivo.

[0040] Figure 13 NALM-6 cells labeled with luciferase, along with unlimited T cells containing or without anti-CD19 CAR, were injected into NSG mice with + / - IL-15. Antitumor efficacy was assessed by BLI (left) and survival (right). Results showed that anti-CD19 unlimited CAR T cells possessed in vivo antitumor efficacy.

[0041] Figure 14 Antigen-specific unlimited T cells. The specificity of HLA-A2-derived antigens against infectious diseases and tumor-associated antigens was tested using HLA-A2 tetramers with known CD8 T-cell epitopes. + Unlimited T cells from donors. Data show the presence of antigen-specific T cells among unlimited T cells, which recognize microbes and tumor-associated antigens via their endogenous TCRs.

[0042] Figure 15Generation of EBV-specific unlimited T cells. On day 0, peripheral blood mononuclear cells from healthy donors (HLA-A2+ donors) were stimulated with a pool of HLA-A2-binding EBV peptides, and CD137-positive T cells were sorted by flow cytometry after 24 hours and used to generate unlimited T cells via transduction of BCL6 and Bcl-xL as previously described. After 7 weeks of culture, tetramer-positive cells were enriched with magnetic beads, and then the enriched cells were cultured for another 6 weeks and stained for CD8 and BMLF1-HLA-A2 tetramers (specific to the HLA-A2-binding peptide (GLCTLVAML) derived from the EBV-BMLF1 protein). These results suggest that enriched microbe or tumor antigen-specific unlimited CD4 or CD8 T cell populations can be generated using the described method.

[0043] Figure 16 Unlimited αβ or γδ T cells were generated using the BCL6 and BCL2L1 genes under the control of a tet-off safety switch. The growth rate of unlimited T cells with IL-2 is shown in the absence (left) and presence (right) of 1 μg / mL doxycycline (Dox). The results suggest that unlimited T cells maintain their growth rate in the absence of doxycycline but cease proliferation and undergo gradual cell death in the presence of doxycycline. A similar tet-off safety switch could also be used to control the incorporation of IL-2 or IL-15 cytokine genes into unlimited T cells.

[0044] Figure 17 Unlimited T cells with a tet-off safety switch were cultured with IL-2 in the presence or absence of progressively increasing concentrations of doxycycline (Dox), and the cells in the culture were imaged by optical microscopy. Cells were also stained after 2 weeks to assess CD25 expression by flow cytometry. Optical microscopy imaging revealed that unlimited T cells gradually decreased in size with increasing concentrations of doxycycline, along with a reduction in the number of cells with proliferating clusters. Furthermore, CD25 expression was significantly reduced in the presence of doxycycline.

[0045] Figure 18 Unlimited T cells with a tet-off safety switch were cultured with IL-2 in the presence or absence of 1 μg / mL doxycycline (Dox), and the cells were stained after 2 weeks to assess the surface markers shown by flow cytometry. PD-1 expression was significantly increased in the presence of doxycycline.

[0046] Figure 19Cytokine production via unlimited T cells. Unlimited T cells (CD8+) with or without anti-CD19 CAR expression were co-cultured with NALM-6 tumor cells at a 5:1 effector:target ratio. Cytokine levels were measured in the supernatant after 3 days. Data represent results from unlimited T cells derived from three different healthy donors. Results showed that unlimited T cells with anti-CD19 CAR (but not without) produced significant amounts of IL-2, GM-CSF, IFNγ, IL-5, and IL-17 in response to NALM-6 tumor cells. Production of TNFα, IL-4, IL-6, IL-10, or IL-13 by anti-CD19 unlimited CAR T cells in response to tumor cells was minimal or not significantly different from that of unlimited T cells without CAR expression. However, we observed that unlimited 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 displayed).

[0047] Figure 20 Lysis of unlimited CAR-T cells via antibody-dependent cell-mediated cytotoxicity (ADCC) using cetuximab. Unlimited T cells expressing anti-CD19 CAR and tEGFR were labeled with CFSE and co-cultured in duplicate with cetuximab or rituximab at 5 μg / mL in the presence of the effector:target ratio shown, with or without NK cells derived from a healthy donor. After 5 hours, the absolute number of unlimited T cells in each well was determined by flow cytometry using counting beads, and the percentage decrease in the number of unlimited T cells compared to T cells alone was calculated and shown in the figure. The percentage decrease in T cells with cetuximab or rituximab in the absence of NK cells was <5%.

[0048] Figures 21A-21CGeneration of unlimited T cells with BCL6 and BCL2L1 genes or BCL6 and BIRC5 (survival protein) genes, a Tet-off safety switch, and IL-15. (Fig. 21A) Design of lentiviral constructs with BCL6 and BCL2L1 genes or BCL6 and BIRC5 genes, a Tet-off safety switch, and IL-15 genes. (Fig. 21B) Human T cells were transduced via lentiviral method using the constructs shown in Plate A and cultured in the presence of IL-2. The growth rate of T cells generated by both methods during in vitro culture under similar conditions was measured after 12 weeks. (Fig. 21C) Unlimited T cells were generated from two donors using the lentiviral constructs containing BCL6 and BCL2L1 genes shown in Plate A and cultured with IL-2 in the presence or absence of 1 μg / mL doxycycline. The cells grew exponentially in the absence of doxycycline but ceased proliferation and underwent gradual cell death in the presence of doxycycline.

[0049] Figure 22 Example of a construct comprising BCL6 and Bcl-xl (L5x(MSCV-BCL6-P2A-BCL-xl-T2A-rtTA)). The structure includes at least wild-type BCL-6 separated from BCL-xL by a P2A element, and BCL-xL separated from rtTA (Tet on trans-activator) by a T2A element.

[0050] Figure 23 This includes illustrated examples of specific embodiments, including at least one for expressing BCL6. Some embodiments include any type of shRNA, including, for example, shRNA targeting cysteine ​​9 or BAK.

[0051] Description of illustrative implementation schemes

[0052] Ectopic expression of the human telomerase reverse transcriptase (hTERT) gene has previously been reported to immortalize normal T cells (Hooijberg et al., 2000). However, it has been observed that overexpression of hTERT alone is insufficient for T lymphocyte immortalization. In fact, T cells generated in this manner cease proliferation after some time (Migliaccio et al., 2000). This study suggests that expression of BCL6 in normal NK or T cells may halt their differentiation, and that expression of genes promoting cell survival (e.g., anti-apoptotic BCL-2 family genes, such as BCL2L1 encoding the Bcl-xL protein) may significantly extend their lifespan, potentially enabling immortalization while maintaining their essential functions.

[0053] Embodiments of this disclosure relate to compositions, production, and uses of cells having a significantly increased lifespan compared to cells lacking the modifications covered herein. In particular embodiments, the cells encode heterologous BCL6 and one or more survival-promoting genes (or anti-apoptotic genes or genes that promote cell survival), including any gene whose gene product has anti-apoptotic function. As an example, the survival-promoting gene may be any BCL-2 family gene, including BCL-xL, BCL-2, MCL-1, or survival protein, only as an example. Additionally or alternatively, the cells have expression inhibition or knockout 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 combinations thereof). In such examples, the DNA fragment used to knock down or eliminate one or more caspase genes can be an shRNA expression cassette. These caspase genes can also be knocked out using gene editing methods (CRISPR, TALEN, zinc finger methods, etc.). Therefore, in a particular embodiment, the immune cells, in addition to BCL6 overexpression or in addition to heterologous BCL6, contain caspase knockout to generate an unlimited number of immune cells. The cells may have one or more survival-promoting genes (or anti-apoptotic genes or genes promoting cell survival), and may also have one or more caspase genes knocked down or eliminated, in particular cases.

[0054] In some embodiments, this disclosure provides a method for generating an unlimited number of immune cells with a significantly increased lifespan and the ability to rapidly grow to large numbers, for example, for adoptive immunotherapy. This method provides unlimited immune cells with the ability to expand indefinitely through a single transduction in at least some cases. This method is very inexpensive and can generate an unlimited number of immune cells within a short timeframe (e.g., one month or longer).

[0055] The platform and system covered herein can be used to generate an unlimited number of immune cells, such as an unlimited number of T cells, including TCRαβ and TCRγδ T cells. This method provides an unrestricted source of human T cells, which can be used as is or can be further genetically modified to generate desired cells, including readily available chimeric antigen receptor (CAR) T cells or T cells transduced via T cell receptors (TCRs). In particular embodiments, the cells are used to treat or prevent cancer and other diseases, including infectious and inflammatory conditions. As an 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 diseases, graft-versus-host disease, or combinations thereof.

[0056] As an example, the studies covered herein demonstrate that transducing anti-CD19 CARs into unlimited T cells generates “anti-CD19 unlimited CAR T cells” (CD19 inCART) and specifically modifies them to target human B-cell tumors. These CD19 unlimited CAR T cells can serve as a source for generating an unlimited number of antigen receptor-modified T cells (e.g., CAR T cells) after only one transduction and exhibit significant cytotoxicity against human B-cell lymphoma cell lines. This disclosure provides off-the-shelf immunocellular therapy platforms and systems that can generate an unlimited number of immune cells and can significantly reduce the cost and production time of adoptive immunocellular therapy by rationalizing the preparation process. Specific embodiments allow for the generation of unlimited cells by expressing BCL6 and one or more survival-promoting genes (or anti-apoptotic genes or genes promoting cell survival), which function as off-the-shelf cells for further manipulation for adoptive cell therapy, such as further manipulation by incorporating a modified target antigen receptor (e.g., tailored for a specific cancer). The existing cells may also already include one or more safety switches (including, for example, inducible systems and elimination genes, such as truncated EGFR (as an example, 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 the steps to adapt the cells to have the desired properties.

[0057] I. Definition

[0058] As used herein, with respect to a specified component, "substantially free" means that none of the specified components is intentionally formulated into the composition and / or exists only as a contaminant or in trace amounts. Therefore, the total amount of the specified component due to any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, it is a composition in which the amount of the specified component cannot be detected by standard analytical methods.

[0059] As used herein, “a” or “an” may mean one or more species. As used in the claims, when used in conjunction with the word “comprising”, the word “a” or “an” may mean one or more species. Some embodiments of this disclosure may consist of or substantially consist of one or more elements, method steps, and / or methods of this disclosure. It is contemplated that any method or composition described herein may be practiced in relation to any other method or composition described herein, and different embodiments may be combined.

[0060] The term “or” is used in the claims to mean “and / or” unless explicitly stated otherwise, referring only to the alternatives or the alternatives are mutually exclusive, although this disclosure supports the definition of referring only to the alternatives and “and / or”. For example, “x, y, and / or z” can mean “x” alone, “y” alone, “z” alone, “x, y, and z”, “(x and y) or z”, “x or (y and z)”, or “x or y or z”. In particular, it is considered that x, y, or z can be specifically excluded from the embodiments. As used herein, “another” can mean at least a second or more. The terms “about,” “substantially,” and “approximately” generally mean the stated value ±5%.

[0061] Throughout this specification, unless the context otherwise requires, the words “comprising,” “including,” and “containing” will be understood to imply inclusion of the described steps or elements or groups of steps or elements, but not to exclude any other steps or elements or groups of steps or elements. “consisting of” means including and limited to whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are necessary or mandatory, and no other elements may be present. “Substantially consisting of” means including any elements listed after this phrase, and limited to other elements that do not interfere with or contribute to the activity or effect of the listed elements as detailed in this disclosure. Thus, the phrase “substantially consisting of” indicates that the listed elements are necessary or mandatory, but other elements are optional and may be present or absent depending on whether they affect the activity or effect of the listed elements.

[0062] Throughout this specification, references to "an embodiment," "(a) embodiment," "(a) particular embodiment," "(a) related embodiment," "a certain embodiment," "(a) additional embodiment," or "(a) further embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment of the invention. Therefore, the appearance of the foregoing phrases in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, the particular feature, structure, or characteristic may be combined in one or more embodiments in any suitable manner.

[0063] "Immune disorders," "immune-related disorders," or "immune-mediated disorders" refer to disorders in which the immune response plays a key role in the development or progression of the disease. Immune-mediated disorders include autoimmune disorders, allogeneic graft rejection, graft-versus-host disease, and inflammatory and allergic conditions.

[0064] An “immune response” is the response of cells of the immune system (e.g., B cells, or T cells, or innate immune cells) to a stimulus. In one embodiment, the response is specific to a particular antigen (“antigen-specific response”).

[0065] "Autoimmune disease" refers to a condition in which the immune system mounts an immune response (e.g., a B cell or T cell response) against antigens that are part of the normal host (i.e., autoantigens), resulting in damage to tissues. Autoantigens can originate from host cells or from symbiotic organisms such as microorganisms (called symbionts) that normally colonize mucosal surfaces.

[0066] "Treatment" of a disease or condition refers to the implementation of a program of treatment that may include administering one or more medications to a patient in an effort to reduce the signs or symptoms of the disease. Desired therapeutic effects include slowing the rate of disease progression, improving or alleviating the disease state, and achieving a reduced or improved prognosis. Relief may occur before or after the signs or symptoms of the disease or condition appear. Therefore, "treatment" may include "prevention" of a disease or undesirable condition. Furthermore, "treatment" does not require complete reduction of signs or symptoms, does not require a cure, and particularly includes programs that have only a marginal effect on the patient.

[0067] As used throughout this application, the terms "therapeutic benefit" or "therapeutic effectiveness" mean anything that promotes or enhances the well-being of a subject with respect to the medical treatment of the condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of the disease. For example, cancer treatment may involve, for instance, a reduction in tumor size, a reduction in tumor invasiveness, a reduction in the rate of cancer growth, or prevention of metastasis. Cancer treatment may also refer to prolonging the survival of a subject with cancer.

[0068] "Subject," "patient," and "individual" can be used interchangeably and can refer to humans or non-humans, such as primates, mammals, and vertebrates. In a particular embodiment, the subject is a human. The subject can be any biological or animal subject that is the target of a method or material, including mammals such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), domestic pets (e.g., dogs, cats, and rodents), horses, and genetically modified non-human animals. The subject can be a patient who, for example, has or is suspected of having a disease (which can be referred to as a medical condition), such as one or more infectious diseases, one or more genetic conditions, one or more cancers, or any combination thereof. As used herein, a "subject" or "individual" may or may not reside in a medical facility and may be treated as an outpatient at a medical facility. The individual may be receiving one or more medical compositions via the Internet. Individuals can include humans or non-human animals of any age, and therefore include adults and juveniles (e.g., children) and infants, and include intrauterine individuals. Subjects may or may not have a need for medical treatment; individuals may be part of an experiment (whether clinical or supporting basic scientific research) voluntarily or involuntarily.

[0069] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that, when administered to animals such as humans (where appropriate), do not produce adverse, allergic, or other troublesome reactions. In light of this disclosure, the preparation of pharmaceutical compositions comprising antibodies or other active ingredients will be known to those skilled in the art. Furthermore, for animal (e.g., human) administration, it will be understood that the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA's Biostandards Agency.

[0070] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral carriers such as sodium chloride, Ringer's glucose solution, 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, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, liquids, and nutrient supplements, as well as similar materials and combinations thereof, as will be known to those skilled in the art. The pH and precise concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0071] II. Unlimited Immune Cells

[0072] Some embodiments of this disclosure relate to immune cells modified to express one or more genes. The expression of said one or more genes directly or indirectly results in an increased lifespan of said cells compared to cells lacking the expression of said one or more genes. In particular embodiments, the cells are manipulated to express said one or more genes, including one or more heterologous genes. In other cases, the cells are manipulated to have upregulation of the expression of said one or more genes endogenous to the cell, for example by manipulating one or more regulatory elements of said one or more genes endogenous to the cell.

[0073] In a particular embodiment, immune cells are manipulated to express BCL6 and one or more pro-survival genes, anti-apoptotic genes, or genes that promote cell survival (and there may be or may not be overlap among genes classified as pro-survival, anti-apoptotic, or cell survival-promoting). As used herein, the pro-survival gene refers to a nucleic acid polymer capable of exerting an anti-apoptotic function or promoting survival through any mechanism. Nucleic acid polymers capable of exerting an anti-apoptotic function can be one or more genes in the Bcl2 family, such as BCL-xL, BCL-2, MCL-1, Bcl-w, Bfl-1, BCL-B, etc. Nucleic acid polymers capable of exerting an anti-apoptotic function can be one or more genes in the inhibitor of apoptosis (IAP) family, such as XIAP, c-IAP1, c-IAP2, NAIP, and survival proteins, etc. Nucleic acid polymers with anti-apoptotic functions can 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 polymers used for knockdown or knockout can be shRNA expression cassettes, or these caspase genes can be knocked out using gene editing methods (CRISPR, TALEN, zinc finger methods, etc.). Nucleic acid polymers with anti-apoptotic functions can also 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 that can exert anti-apoptotic functions can have anti-apoptotic effects, such as 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.

[0074] Unlimited T cells can be generated using either wild-type or mutant BCL6. The inventors determined that unlimited T cells can be generated using either wild-type BCL6 or mutant BCL6 (with a single specific nucleotide difference – codon CCT (encoding proline / P) for amino acid position 395 in wild-type BCL6 and codon CTT (encoding leucine / L) for amino acid position 395 in mutant BCL6). The nucleotide and amino acid sequences of these two BCL6 genes are shown below (mutation points in the wild-type sequence are underlined).

[0075] wild-type BCL6 aa sequence:

[0076] MASPADSCIQFTRHASDVLLNLNRLRRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCESRAFAPSLYSGLSTPPASYSM YSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEG P EQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQ ASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC(SEQ ID NO:1).

[0077] The nucleotide sequence of wild-type BCL6 (codons at mutation sites in the wild-type sequence are underlined):

[0078] cCt gagcaggctgagctgggccgcctttccccacgagcctacacggccccacctgcctgccagccacccatggagcctgagaaccttgacctccagtccccaaccaagctgagtgccagcggggaggactccaccatcccacaagccagccggctcaataacatcgttaacaggtccatgacgggctctccccgcagcagcagcgagagccactcaccactctacatgcaccccccgaagtgcacgtcctgcggctctcagtccccacagcatgcagagatgtgcctccacaccgctggccccacgttccctgaggagatgggagagacccagtctgagtactcagattctagctgtgagaacggggccttcttctgcaatgagtgtgactgccgcttctctgaggaggcctcactcaagaggcacacgctgcagacccacagtgacaaaccctacaagtgtgaccgctgccaggcctccttccgctacaagggcaacctcgccagccacaagaccgtccataccggtgagaaaccctatcgttgcaacatctgtggggcccagttcaaccggccagccaacctgaaaacccacactcgaattcactctggagagaagccctacaaatgcgaaacctgcggagccagatttgtacaggtggcccacctccgtgcccatgtgcttatccacactggtgagaagccctatccctgtgaaatctgtggcacccgtttccggcaccttcagactctgaagagccacctgcgaatccacacaggagagaaaccttaccattgtgagaagtgtaacctgcatttccgtcacaaaagccagctgcgacttcacttgcgccagaagcatggcgccatcaccaacaccaaggtgcaataccgcgtgtcagccactgacctgcctccggagctccccaaagcctgc(SEQ ID NO:2)。

[0079] The aa sequence of mutant BCL6 (leucine mutation is underlined):

[0080] MASPADSCIQFTRHASDVLLNLNRLRRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCESRAFAPSLYSGLSTPPASYSM YSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEG L EQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQ ASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC(SEQ ID NO:3).

[0081] The nucleotide sequence of mutant BCL6 (codons for leucine are underlined):

[0082] cTt gagcaggctgagctgggccgcctttccccacgagcctacacggccccacctgcctgccagccacccatggagcctgagaaccttgacctccagtccccaaccaagctgagtgccagcggggaggactccaccatcccacaagccagccggctcaataacatcgttaacaggtccatgacgggctctccccgcagcagcagcgagagccactcaccactctacatgcaccccccgaagtgcacgtcctgcggctctcagtccccacagcatgcagagatgtgcctccacaccgctggccccacgttccctgaggagatgggagagacccagtctgagtactcagattctagctgtgagaacggggccttcttctgcaatgagtgtgactgccgcttctctgaggaggcctcactcaagaggcacacgctgcagacccacagtgacaaaccctacaagtgtgaccgctgccaggcctccttccgctacaagggcaacctcgccagccacaagaccgtccataccggtgagaaaccctatcgttgcaacatctgtggggcccagttcaaccggccagccaacctgaaaacccacactcgaattcactctggagagaagccctacaaatgcgaaacctgcggagccagatttgtacaggtggcccacctccgtgcccatgtgcttatccacactggtgagaagccctatccctgtgaaatctgtggcacccgtttccggcaccttcagactctgaagagccacctgcgaatccacacaggagagaaaccttaccattgtgagaagtgtaacctgcatttccgtcacaaaagccagctgcgacttcacttgcgccagaagcatggcgccatcaccaacaccaaggtgcaataccgcgtgtcagccactgacctgcctccggagctccccaaagcctgc(SEQ ID NO:4)

[0083] The immune cells can be any type of immune cell, including T cells (e.g., regulatory T cells, CD4+). + T cells, CD8 + T cells, αβT cells, γδT cells, or mixtures thereof; NK cells, invariant NKT cells, NKT cells, innate lymphoid cells, or mixtures thereof. The immune cells may be virus-specific, expressing CAR, and / or expressing TCR. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells (DCs), mast cells, eosinophils, and / or basophils. Methods for generating and modifying the immune cells, as well as methods for using and administering the cells for adoptive cell therapy, are also provided herein, in which case the cells may be autologous or allogeneic. Thus, the immune cells can be used as immunotherapy, for example, for targeting cancer cells. These immune cells can be used for treatment as a single cell type or as a combination of multiple immune cell types. In particular embodiments, the immune cells are CD3+, CD4+, CD8+, CD16+, or mixtures thereof.

[0084] The immune cells can be isolated from a subject, particularly a human subject. The immune cells can be obtained from a subject intended for a specific disease or condition, such as 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 within the subject, including but not limited to blood, umbilical cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells can be used directly or stored for a period of time, such as by freezing.

[0085] The immune cells can be enriched / purified from any tissue in which they are located, including but not limited to blood (including blood collected through blood banks or cord blood banks), spleen, bone marrow, tissue removed and / or exposed during surgical procedures, and tissue obtained via biopsy procedures. The tissue / organ from which the immune cells are enriched, isolated, and / or purified can be isolated from living and non-living subjects, wherein the non-living subject is an organ donor. In a particular embodiment, the immune cells are isolated from blood, such as peripheral blood or cord blood. In some aspects, immune cells isolated from cord blood have enhanced immunomodulatory capacity, for example, as measured by CD4- or CD8-positive T cell suppression. In a particular aspect, the immune cells are isolated from pooled blood, particularly pooled cord blood, for enhanced immunomodulatory capacity. The pooled blood may be from two or more sources, such as three, four, five, six, seven, eight, nine, ten, or more sources (e.g., donor subjects).

[0086] The immune cell population can be obtained from a subject requiring treatment or suffering from a disease associated with reduced immune cell activity. Therefore, the cells will be autologous for the subject requiring treatment. Alternatively, the immune cell population can be obtained from a donor, such as a partially or fully tissue-matched donor or a completely non-tissue-matched donor. The immune cell population can be harvested from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which the immune cells are located in the subject or donor. The immune cells can be isolated from a collection of the subject and / or donor, such as from a collection of umbilical cord blood.

[0087] When the immune cell population is obtained from a donor different from the subject, the donor may be allogeneic, provided that the obtained cells are compatible with the subject so that they can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible.

[0088] AT cells

[0089] In some embodiments, the immune cells are T cells. Several fundamental methods for the derivation, activation, and expansion of functional anti-tumor effector cells have been described over the past two decades. These include: autologous cells, such as tumor-infiltrating lymphocytes (TILs); ex vivo activated T cells, obtained by using autologous dendritic cells (DCs) or PBMCs, lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells isolated by capturing target cell membranes; allogeneic cells naturally expressing anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells genetically reprogrammed or “modified” to express tumor-reactive TCRs or chimeric TCR molecules exhibiting antibody-like tumor recognition capabilities, termed “T-antibodies (T-body).” These methods have led to numerous protocols for T cell preparation and immunization, which can be employed in the methods described herein.

[0090] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphatic organs. In some aspects, the cells are human cells. The cells are typically primary cells, such as those directly isolated from a subject and / or isolated from a subject and frozen. In some embodiments, the cells include one or more T cell subclasses or other cell types, such as the entire T cell population, CD4+, etc. + Cells, CD8 +Cells and their subpopulations, for example, those defined by the following aspects: function, activation state, maturity, potential for differentiation, expansion, recycling, localization, and / or persistence, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, biomarker or cytokine secretion profile, and / or degree of differentiation. Regarding the subject to be treated, the cells may be allogeneic and / or autologous. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and / or specifically pluripotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from the subject, preparing, processing, culturing, and / or modifying them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0091] In T cell subtypes and subsets (e.g., CD4) + and / or CD8 + T cells include naive T cells (T2). N ) cells, effector T cells (T cells) EFF ), memory T cells and their subtypes, such as stem cell memory T cells (TSCs). M ), central memory T cells (TC) M ), effector memory T cells (T EM ) or terminal differentiation effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and γ / δ T cells.

[0092] In some implementations, one or more cells in the T cell population are enriched or depleted that are positive for a specific marker (e.g., a surface marker) or negative for a specific marker. In some cases, such markers are those that are absent or expressed at relatively low levels in some T cell populations (e.g., non-memory cells), but present or expressed at relatively higher levels in some other T cell populations (e.g., memory cells).

[0093] In some implementations, T cells are separated from PBMC samples by negative selection of markers (e.g., CD14) expressed on non-T cells (e.g., B cells, monocytes, or other leukocytes). In some aspects, CD4 is used. + or CD8 + Choose the steps to separate CD4 + Helper T cells and CD8+ Cytotoxic T cells. These CD4+ cells can be classified by positive or negative selection for markers expressed or expressed at relatively high levels on one or more naive, memory, and / or effector T cell subsets. + and CD8 + The group was further subdivided into subgroups.

[0094] In some implementations, CD8 is made + T cells further enrich or deplete naive, central memory, effector memory, and / or central memory stem cells, for example, through positive or negative selection based on surface antigens associated with their respective subsets. In some embodiments, selection is performed on central memory T(T) cells. CM Enrichment of memory cells or stem cells to increase potency, such as to improve long-term survival, expansion and / or translocation after application, is particularly robust in some respects in this subpopulation.

[0095] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from the patient, and a single-cell suspension is obtained. The single-cell suspension can be obtained in any suitable manner, such as mechanically (by depolymerizing the tumor, for example using gentleMACS). TM Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single-cell suspensions of tumor enzymatic digests are cultured in interleukin-2 (IL-2) or other growth factors.

[0096] Cultured T cells can be pooled and rapidly expanded. Rapid expansion provides at least a 50-fold (e.g., 50, 60, 70, 80, 90, or 100-fold or greater) increase in the number of antigen-specific T cells over a period of approximately 10 to approximately 14 days. More preferably, rapid expansion provides at least a 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900, or greater) increase over a period of approximately 10 to approximately 14 days.

[0097] Expansion can be accomplished by any of the many methods known in the art. For example, T cells can be rapidly expanded by using nonspecific T-cell receptor stimulation in the presence of fed lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15) (wherein IL-2 is preferred). The nonspecific T-cell receptor stimulation may comprise approximately 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (from Ortho- Raritan, NJ (available). Alternatively, T cells can be rapidly expanded by stimulating peripheral blood mononuclear cells (PBMCs) in vitro with one or more antigens of the cancer (including its antigenic portions, such as epitopes or cells) (which may optionally be expressed from a vector) (e.g., human leukocyte antigen A2 (HLA-A2) binding peptides or peptides bound to other MHC class I or II molecules) in the presence of T-cell growth factors such as 300 IU / ml IL-2 or IL-15 (wherein IL-2 is preferred). The in vitro-induced T cells are then rapidly expanded by restimulating them with the same antigen of the cancer pulsed onto antigen-presenting cells expressing HLA-A2 or expressing other HLA molecules. In vitro-induced T cells can also be expanded in the absence of antigen-presenting cells.

[0098] The autologous T cells can be modified to express T cell growth or 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*, 3rd edition, 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 particular, the modified T cells express T cell growth factors at high levels. T cell growth factor coding sequences (e.g., the coding sequence for IL-12) are readily available in the art, and operative linkage of these sequences to promoters promotes high-level expression.

[0099] B. NK cells

[0100] In some embodiments, the immune cells are natural killer (NK) cells. NK cells are a subset of lymphocytes that possess spontaneous cytotoxicity against a wide variety of 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. NK cells can be detected by specific surface markers (e.g., CD16, CD56, and / or CD8 in humans). NK cells do not express T cell antigen receptors, the pan-T marker CD3, or surface immunoglobulin B cell receptors.

[0101] In some embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte removal products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, tissue, or umbilical cord blood using methods well known in the art.

[0102] C.NKT cells

[0103] Natural killer T (NKT) cells are a heterogeneous group of T cells that share characteristics with both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds to both self- and exogenous lipids and glycolipids. They constitute only approximately 0.1% of all peripheral blood T cells. NKT cells are a subclass of T cells that co-express the αβ T-cell receptor but also express a wide variety of molecular markers typically associated with NK cells (e.g., NK1.1). Invariant natural killer T (iNKT) cells express high levels of the transcriptional regulator promyelocytic leukemia zinc finger and depend on this regulator for their development. Currently, five major distinct iNKT cell subclasses exist. These subclasses produce different sets of cytokines upon activation. Isotypes iNKT1, iNKT2, and iNKT17 reflect Th cell subclasses in cytokine production. Additionally, there are subtypes specialized in T follicle helper-like functions and IL-10-dependent regulatory functions.

[0104] D. Congenital lymphoid cells

[0105] Innate lymphoid cells (ILCs) are a group of innate immune cells derived from common lymphoid progenitor cells (CLPs) and belonging to the lymphoid lineage. These cells are defined by the absence of antigen-specific B or T cell receptors due to the lack of recombinant activation genes (RAGs). ILCs do not express myeloid or dendritic cell markers. They play a role in protective immunity and the regulation of homeostasis and inflammation; therefore, dysregulation of ILCs can lead to immunopathological states such as allergic reactions, bronchial asthma, and autoimmune diseases. ILCs can be classified based on the cytokines they produce and the transcription factors that regulate their development and function.

[0106] III. Unlimited production of immune cells

[0107] In some aspects, this disclosure provides methods for increasing the lifespan of immune cells by overexpressing BCL6 and one or more pro-survival genes or anti-apoptotic genes or genes that promote cell survival (including one or more anti-apoptotic BCL-2 family genes, such as Bxl-xL). The gene expression can be achieved using conventional molecular biology methods, such as by cloning the coding sequences of BCL6 and the 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 vector to immune cells. Alternatively, the gene expression can be achieved by specifically transcribing the mRNA of BCL6 and the anti-apoptotic BCL-2 family genes in immune cells using CRISPR or other transposases (as an example). The expression of BCL6 and / or the anti-apoptotic BCL-2 family member (e.g., Bcl-xL) can be regulated, including through constitutive or inducible means. In some cases, the expression of BCL6 and / or the anti-apoptotic BCL-2 family members can be regulated by a first type of expression regulation (e.g., constitutive), and the expression of one or more other genes in the system (e.g., on the same or different vectors) can be regulated in the same way (e.g., constitutive) or differently (e.g., inducible). In particular, BCL6-BCL-xL is regulated via a tet-off or tet-on regulatory mechanism.

[0108] In one exemplary method, the coding sequences of the BCL6 and Bcl-xL genes (by way of example only) can be linked together, but separated by elements that allow for the eventual generation of separate BCL6 and Bcl-xL molecules. For example, the coding sequences of the BCL6 and Bcl-xL genes can be linked together, but separated by a T2A sequence to generate an open reading frame that can simultaneously express the BCL6 and Bcl-xL genes. This BCL6-T2A-Bcl-xL open reading frame can be cloned into a vector, such as a lentiviral vector. The immune cells (e.g., T cells) can then be transduced via this viral vector, for example in the presence of IL-2 and / or IL-15. This method can generate a T cell line called “unlimited T cells” from healthy donor T cells, which can proliferate in the presence of recombinant human IL-2 and / or IL-15. In some cases, the cells are generated in the presence of IL-2 and / or IL-15, and the cells themselves also express heterologous IL-2 and / or IL-15, although in other cases, only one of these parameters is used.

[0109] Here is an example of a self-cutting sequence:

[0110] T2A(GSG)EGRGSLLTCGDVEENPGP(SEQ ID NO:5)

[0111] P2A(GSG)ATNFSLLKQAGDVEENPGP(SEQ ID NO:6)

[0112] E2A(GSG)QCTNYALLKLAGDVESNPGP(SEQ ID NO:7)

[0113] F2A(GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:8).

[0114] In other cases, the IRES element is used instead of the 2A sequence.

[0115] In some embodiments, the cells are modified to express the BCL6-2A-BCLxL sequence (SEQ ID NO:9), which comprises human BCL6, 2A self-cleaving peptide and BCL-xl coding sequence.

[0116]

[0117] Another example of an expression construct containing BCL6 and Bcl-xL is shown below, where the part with a single underscore represents BCL6, the part without an underscore represents P2A, and the part with a double underscore represents Bcl-xL:

[0118]

[0119] An example of a construct containing BCL6 and Bcl-xl (L5x(MSCV-BCL6-P2A-BCL-xl-T2A-rtTA); see Figure 21) is shown below. The overall structure is as follows: NNNN-CMV promoter NN-HIV-LTR-HIV1_psi pack-spacer-RRE-spacer-cPPT-MSCV promoter-BCL-6WT-P2A-BCL-xL-T2A-rtTA-WPRE-U3PPT-HIV-LTR-bGHpA-SV40 Origin of Replication-plasmid Origin of Replication-ampicillin Resistance Gene-AmpR_promoter—NNNN. The specific sequences of specific domains of the constructs below (and in Figure 21) are depicted immediately following SEQ ID NO:11 below:

[0120]

[0121] CMV promoter

[0122] ACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGC(SEQ ID NO:61).

[0123] HIV LTR

[0124] GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA(SEQ ID NO:62).

[0125] HIV1 psi packaging signal

[0126] TGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAG(SEQ ID NO:63).

[0127] RRE

[0128] AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCT(SEQ ID NO:64).

[0129] cPPT

[0130] AAAAGAAAAGGGGGGA(SEQ ID NO:65).

[0131] MSCV promoter

[0132] aatgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcggcgcgccagtcctccgatagactgcgtcgcccgggtacccgtattcccaataaagcctcttgctgtttgcatccgaatcgtggactcgctgatccttgggagggtctcctcagattgattgactgcccacctcgggggtctttcat(SEQ IDNO:66)。

[0133] BCL-6 WT

[0134]

[0135] P2A

[0136] GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT(SEQ ID NO:68)。

[0137] BCL-xL

[0138] (SEQ ID NO:69)。

[0139] T2A

[0140] GGCAGTggcgagggtagaggttctctcctcacttgtggtgatgttgaagaaaaccctggtcca(SEQID NO:70)。

[0141] rtTA

[0142] atgtctagactggacaagagcaaagtcataaacggagctctggaattactcaatggtgtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatgctggacaggcatcatacccacttctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccgctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgagacaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaacagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaaGGTgA(SEQ ID NO:71)。

[0143] WPRE

[0144] TCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCA(SEQ ID NO:72)。

[0145] U3PPT

[0146] AAAAGAAAAGGGGGGA(SEQ ID NO:73)。

[0147] -HIV-LTR

[0148] GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA(SEQ ID NO:74)。

[0149] bGH pA

[0150] CGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATG(SEQ ID NO:75).

[0151] SV40 replication origin

[0152] Atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcc(SEQ IDNO:76).

[0153] Plasmid replication origin

[0154] TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA(SEQ ID NO:77).

[0155] Ampicillin resistance gene

[0156] TTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT(SEQ ID NO:78).

[0157] AmpR promoter

[0158] ATTGTCTCATGAGCGGATACATATTTGAA(SEQ ID NO:79).

[0159] In a further aspect, this disclosure provides unlimited immune cells that can be genetically modified to predispose them to target specific organ sites or tumor markers. The unlimited immune cells may express one or more suicide or elimination genes, which can be used to eliminate unlimited immune cells from a patient in the event of a serious adverse event. The unlimited immune cells may express one or more genes (including genes encoding IL-2 and / or IL-15) capable of maintaining or enhancing the proliferation of unlimited T cells for in vivo application. The expression of IL-2 and / or IL-15 may be constitutively expressed or regulated, for example, doxycycline-regulated (Tet-on or Tet-off). The cells can be engineered to express one or more other cytokines, such as IL-7, IL-12, IL-18, IL-21, etc.; one or more chemokine receptors, such as 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, such as CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, etc. 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.

[0160] Unlimited immune cells can be modified to express antigen-specific CARs or TCRs to target tumors or infections. Another strategy for targeting tumors is to modify unlimited T cells to express CARs with Fc receptors on their extracellular domains, allowing them to be used in combination with monoclonal antibodies targeting tumor markers. Additionally, unlimited immune cells can be modified 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 guide the transport of these molecules to target organ sites.

[0161] A further embodiment provides unlimited immune cells with one or more safety switches (e.g., any kind of suicide or elimination gene). In some embodiments, the system may use a truncated human epidermal growth factor receptor (hEGFRt), HSV-TK, SR39 mutant HSV-TK, yeast CD gene, or a mutant CD20 thereof. When using hEGFRt, the gene can give unlimited T cells the property of being recognized and eliminated by an FDA-approved monoclonal antibody (e.g., cetuximab) when they are no longer needed. For example, the gene can act as a safety switch in the event of a serious adverse event following the injection of therapeutic unlimited immune cells. In addition to acting as a safety switch, hEGFRt can also act as a biomarker to enrich CAR-positive cells and track these cells after infusion into a patient.

[0162] An example of a truncated EGFR is shown below, in which domains 1 and 2 of the EGFR have been removed:

[0163] DNA sequence:

[0164]

[0165] Amino acid sequence of truncated EGFR lacking domains 1 and 2: MLLLVTSLLCELPHPAFLRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFG TSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR(SEQ ID NO:13).

[0166] In some embodiments, the fusion protein serving as the safety switch is a fusion of EGFR (domain 3) and HER2 (domain IV) proteins. In such cases, EGFR domain 3 is an antibody-binding domain, and HER2 domain 4 comprises an extracellular spacer and a transmembrane domain. In a particular embodiment, the fusion protein is a molecule separate from the CAR.

[0167] Any one or more genes or expression constructs in the infinite cells may or may not be regulated, for example, via a doxycycline-regulated Tet-on or Tet-off system. An example of a Tet-responsive promoter sequence includes the following Tet-responsive promoter containing seven repeats of the Tet-responsive element:

[0168] gagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgatgtcgagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatc agtgatagagaacgtatgtcgagtttatccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgtatgtcgaggtaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcc(SEQ ID NO:14).

[0169] For the tet system, an example of the DNA sequence for tTA (Tet off) is as follows:

[0170] ATGAGCCGCCTGGATAAGTCCAAAGTGATCAACTCTGCCCTGGAGCTGCTGAATGAAGTGGGCATCGAGGGCCTGACCACACGGAAGCTGGCCCAGAAGCTGGGAGTGGAGCAGCCAACCCTGTACTGGCACGTGAAGAACAAGCGCGCCCTGCTGGACGCCCTGGCCATCGAGATGCTGGATCGGCACCACACACACTTCTGCCCCCTGGAGGGAGAGTCCTGGCAGGATTTCCTGCGGAACAATGCCAAGAGCTTTAGATGTGCACTGCTGTCCCACAGGGACGGAGCAAAGGTGCACCTGGGCACCAGGCCTACAGAGAAGCAGTACGAGACCCTGGAGAACCAGCTGGCCTTCCTGTGCCAGCAGGGCTTTTCTCTGGAGAATGCACTGTATGCACTGAGCGCCGTGGGACACTTCACCCTGGGATGCGTGCTGGAGGACCAGGAGCACCAGGTGGCCAAGGAGGAGAGAGAGACACCCACCACAGATTCCATGCCCCCTCTGCTGAGGCAGGCCATCGAGCTGTTTGACCACCAGGGAGCAGAGCCTGCCTTCCTGTTTGGCCTGGAGCTGATCATCTGCGGCCTGGAGAAGCAGCTGAAGTGTGAGTCTGGAGGACCAGCAGACGCCCTGGACGATTTCGACCTGGATATGCTGCCCGCCGATGCCCTGGACGATTTTGACCTGGATATGCTGCCTGCCGACGCCCTGGACGATCTGGACCTGGATATGCTGCCAGGCacc(SEQID NO:15).

[0171] An example of the amino acid sequence of tTA (Tet off) is as follows:

[0172] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDLDLDMLPG(SEQ ID NO:16).

[0173] An example of the DNA sequence of rtTA (Tet on) is as follows:

[0174] atgtctagactggacaagagcaaagtcataaacggagctctggaattactcaatggtgtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatgctggacaggcatcatacccacttctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccgctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgagacaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaacagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaa(SEQID NO:17).

[0175] An example of the amino acid sequence of rtTA (Tet on) is as follows:

[0176] MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFS LENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG(SEQ ID NO:18).

[0177] In some aspects, the unlimited immune cells can be engineered to express one or more cytokines, including IL-2 and / or IL-15, such as inducible IL-2 and / or IL-15, for example, to maintain or enhance proliferation. However, in particular cases, any cytokines in the system can be constitutively regulated. For example, unlimited immune cells can produce IL-15 and / or IL-2 in the presence of an inducing agent (e.g., doxycycline) to support their own proliferation. By adjusting the dosage of doxycycline, the survival and proliferation of unlimited immune cells can be maintained or regulated in vivo.

[0178] A specific IL-2 sequence can be used. In at least some cases, there are two DNA sequence examples of IL-2, and they both encode the same IL-2 amino acid sequence.

[0179] IL-2 DNA sequence 1:

[0180] ATGTATCGGATGCAACTCCTCAGCTGCATTGCGTTGTCACTCGCACTCGTCACGAACTCTGCACCGACATCTAGTAGTACTAAGAAAACACAGTTGCAACTGGAGCACCTGCTGTTGGATTTGCAAATGATCCTTAACGGGATCAACAACTACAAAAACCCTAAGCTCACACGAATGCTTACTTTCAAGTTTTACATGCCGAAAAAAGCCACAGAGCTGAAGCATCTTCAGTGCCTTGAAGAGGAGCTTAAACCCCTCGAGGAGGTACTGAATCTCGCGCAAAGCAAGAATTTTCATTTGCGGCCCCGGGACCTTATATCAAACATTAACGTGATCGTGTTGGAACTCAAGGGATCAGAGACGACATTTATGTGCGAGTACGCTGACGAGACCGCTACAATCGTAGAGTTTCTCAATAGGTGGATCACGTTTTGCCAAAGCATCATCTCAACGCTC(SEQ ID NO:19).

[0181] IL-2 DNA sequence 2:

[0182] ATGTATAGGATGCAGCTGCTGTCCTGCATCGCCTTGTCCCTGGCCCTTGTGACCAACAGCGCCCCAACCTCCTCCTACCAAAAAAACCCAACTTCAGCTTGAGCATCTCCTCTTGGACCTGCAGATGATCCTGAATGGTATAAACAACTACAAGAACCCCAAGCTGACCCGGATGCTTACATTCAAATTCTATATGCCTAAAAAGGCTACAGAGCTGAAGCACCTGCAGTG CCTGGAAGAGGAGCTGAAGCCACTGGAAGAGGTCCTGAACTTGGCCCAGAGCAAGAACTTTCACCTCAGGCCCAGGGACTTGATAAGCAACATAAATGTAATCGTCCTGGAGCTGAAGGGGTTCTGAAACAACCTTCATGTGTGAGTATGCAGATGAGACCGCTACCATCGTGGAGTTCCTCAACAGATGGATTACATTTTGTCAATCCATCATCAGCACCCTGACATCT(SEQ ID NO:20).

[0183] In some embodiments, a specific IL-2 amino acid sequence is used in said cells:

[0184] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL (SEQ ID NO: 21).

[0185] In some implementations, a specific IL-15 nucleic acid polymer sequence is used in the cells:

[0186] ATGGGCCTGACCTCTCAGCTGCTGCCACCCCTGTTCTTTCTGCTGGCCTGTGCCGGCAATTTCGTGCACGGCGCCAACTGGGTGAATGTGATCTCTGACCTGAAGAAGATCGAGGATCTGATCCAGAGCATGCACATCGACGCCACCCTGTATACAGAGTCCGATGTGCACCCTTCTTGCAAGGTGACAGCCATGAAGTGTTTTCTGCTGGA GCTGCAGGTCATCTCTCTGGAGAGCGGCGACGCCAGCATCCACGATACCGTGGAGAATCTGATCATCCTGGCCAACAAATAGCCTGAGCTCCAACGGCAATGTGACAGAGTCCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCCTTTGTGCACATCGTGCAGATGTTTATCAATACCTCTTGA(SEQ ID NO:22).

[0187] In some embodiments, a specific IL-15 amino acid sequence is used in said cells:

[0188] MGLTSQLLPPLFFLLACAGNFVHGANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQID NO: 23).

[0189] In particular cases, the immune cells contain IL-15 fused to part or all of the IL-15 receptor. In another particular case, the immune cells contain IL-15 fused to the sushi domain of the IL-15 receptor α unit, and an example of its sequence is as follows:

[0190] MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDGGGGSGGGGSGGGGSNWV NVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:24).

[0191] The DNA sequence of IL-15 fused to the sushi domain of the IL-15 receptor α unit:

[0192] (SEQ ID NO:25).

[0193] The unlimited immune cells can be genetically engineered to give them target selectivity by introducing one or more chimeric antigen receptors (CARs) that can recognize specific tumor markers such as CD19, CD20, CD22, and / or mesothelin, and / or T-cell receptors (TCRs), such as TCRs targeting EBV, CMV, or NY-ESO-1. An example is the “anti-CD19 unlimited CAR T cell” (CD19 inCART), mentioned elsewhere in this document. CD19 is expressed in almost all types of B-cell lymphomas or B-cell leukemias and in normal B cells. CD19 inCART is generated by delivering a lentivirus or non-viral vector expressing an anti-CD19 CAR into selected unlimited cells.

[0194] The unlimited immune cells can also be genetically modified to confer additional properties, such as i) resistance to T cell exhaustion by knocking out or downregulating receptors or ligands PD-1, LAG-3, TIM-3, PD-L1, etc.; ii) resistance to immunosuppressive mechanisms, such as by knocking out or downregulating TGF-β receptors; iii) prevention of graft-versus-host disease by knocking out TCRs; iv) improved potency 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 downregulating MHC molecules; or by expressing surface ligands or other surface or intracellular molecules in the unlimited immune cells to suppress or reduce the function of host immune cells.

[0195] The unlimited immune cells can be generated by special methods or under special conditions. For example, in a particular embodiment, during the generation of the unlimited immune cells, the cells may be subjected to one or more special reagents simultaneously with generation, which enhance their potency after generation, at least compared to their potency without exposure to the one or more special reagents. For example, in some cases, IL-2 is used to generate and expand unlimited T cells. In a particular embodiment, one or more different combinations of cytokines (IL-2, IL-7, IL-21, IL-15, IL-12, IL-18, IL-23, IFN-γ, TNF-α, etc.) and / or chemokines can be used to prepare unlimited T cells with a specific phenotype and specific function.

[0196] IV. Genetically modified antigen receptors

[0197] The immune cells disclosed herein may or may not be genetically modified to express one or more antigen receptors, such as one or more modified TCRs and / or one or more CARs. For example, the immune cells may be modified to express CARs and / or TCRs having antigen specificity for cancer antigens or microbial antigens (including pathogenic antigens). Multiple CARs and / or TCRs may be added to the immune cells, for example, those targeting different antigens. In some aspects, the immune cells are modified to express CARs or TCRs by knocking in a CAR or TCR at a repressive gene locus using gene editing methods such as CRISPR / Cas9.

[0198] Suitable modification methods are known in the art. See, for example, Sambrook and Ausubel, above. For instance, the cells can be transduced to express TCRs with antigen specificity for cancer antigens by using transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009.

[0199] Electroporation of RNA encoding full-length TCRα and β (or γ and δ) chains can be used as an alternative to overcome the long-standing problem of autoreactivity caused by the pairing of retroviral-transduced and endogenous TCR chains. Even if such alternative pairings occur in transient transfection strategies, the resulting autoreactive T cells will lose this autoreactivity after some time because the introduced TCRα and β chains are only transiently expressed. When the expression of the introduced TCRα and β chains decreases, only normal autologous T cells remain. This is not the case when a full-length TCR chain is introduced via stable retroviral transduction, which never loses the introduced TCR chain, resulting in persistent autoreactivity in the patient.

[0200] In some embodiments, the cell comprises one or more nucleic acid polymers introduced via genetic modification that encode one or more antigen receptors, and genetically modified products of such nucleic acid polymers. In some embodiments, the nucleic acid polymer is heterologous, i.e., not normally present in the cell or a sample obtained from the cell, such as that obtained from another organism or cell, and is not typically found in the cell being modified and / or the organism from which such cells are derived. In some embodiments, the nucleic acid polymer is not naturally occurring, such as a nucleic acid polymer not found in nature (e.g., chimeric).

[0201] In some embodiments, the CAR includes 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 cells, including specific cancer cells. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of major histocompatibility complex (MHC) molecules.

[0202] Exemplary antigen receptors (including CARs and recombinant TCRs) and methods for modifying said receptors and introducing said receptors into cells, including, for example, those described in International Patent Application Publications Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publications Nos. US2002131960, US2013287748, US20130149337, and U.S. Patent No. 6,450. 1,995, 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 those described in European Patent Application No. EP2537416; and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some aspects, the genetically modified antigen receptor includes the CAR described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO / 2014055668A1.

[0203] A. Chimeric antigen receptor

[0204] In some embodiments, the CAR includes: a) an intracellular signal transduction domain; b) a transmembrane domain; c) an extracellular domain containing an antigen-binding region; and optionally, d) one or more co-stimulatory domains.

[0205] In some embodiments, the modified antigen receptor comprises a CAR, including activating or stimulatory CARs, co-stimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). The CAR typically contains an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some respects via a linker and / or a transmembrane domain. Such molecules typically mimic or approximate signaling via natural antigen receptors, signaling via such receptors and co-stimulatory receptors in combination therewith, and / or signaling via individual co-stimulatory receptors.

[0206] Some embodiments of this disclosure relate to the use of nucleic acid polymers, including nucleic acid polymers encoding polypeptides such as antigen-specific CAR polypeptides, including humanized CARs (hCARs) to reduce immunogenicity, which comprise intracellular signal transduction domains, transmembrane domains, and extracellular domains (containing one or more signal transduction motifs). In some embodiments, the CAR can recognize an epitope contained in a shared space between one or more antigens. In some embodiments, the binding region may comprise a complementarity-determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, that specificity derives from a peptide (e.g., a cytokine) that binds to a receptor.

[0207] The human CAR nucleic acid polymer is considered to be a human gene for enhancing cell immunotherapy for human patients. In a particular embodiment, the invention includes a full-length CAR cDNA or coding region. The antigen-binding region or domain may comprise a V-shaped variable fragment (scFv) derived from a specific human monoclonal antibody (e.g., those described in U.S. Patent 7,109,304, which is incorporated herein by reference). H and V L The fragment is a segment of the chain. The fragment can also be any number of different antigen-binding domains of human antigen-specific antibodies. In a more particular embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage in order to be expressed in human cells.

[0208] The arrangement can be multimeric, such as a double-chain antibody or a multimer. The multimer is most likely formed by cross-pairing variable portions of the light and heavy chains into a double-chain antibody. The hinge portion of the construct can have multiple options, from complete deletion to retaining the first cysteine, to proline substitution instead of serine, to truncation down to the first cysteine. The Fc portion can be deleted. Any stable and / or dimerized protein can be used for this purpose. Only one of the Fc domains can be used, such as the CH2 or CH3 domains from human immunoglobulins. The hinge, CH2, and CH3 regions of human immunoglobulins modified to improve dimerization can also be used. Only the hinge portion of the immunoglobulin can be used. A portion of CD8α or a synthetic molecule can also be used.

[0209] In some embodiments, the CAR nucleic acid comprises partial or complete sequences encoding other co-stimulatory receptors, individually or in combination, such as natural or modified extracellular domains, transmembrane domains, and intracellular signaling domains of specific molecules (e.g., CD28). Other co-stimulatory domains include, but are not limited to, one or more of the following: 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 signaling induced by CD3ζ, the additional signaling provided by the co-stimulatory receptors inserted into the CAR is important for the complete activation of NK cells and can help improve the in vivo persistence and therapeutic success of adoptive immunotherapy.

[0210] In some embodiments, a CAR is constructed that is specific for a particular antigen (or biomarker or ligand), such as an antigen expressed in a specific cell type to be targeted by adoptive therapy, such as a cancer biomarker, and / or an antigen intended to induce a mitigating response, such as an antigen expressed in normal or disease-free cell types. Therefore, the CAR typically contains one or more antigen-binding molecules in its extracellular portion, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR contains the antigen-binding portion of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0211] In some embodiments of the chimeric antigen receptor, 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 pathogen-specific antigen-binding domain. The antigen includes carbohydrate antigens recognized by pattern recognition receptors (e.g., Dectin-1). The tumor-associated antigen can be of any kind, as long as it is expressed on the cell surface of tumor cells. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigens, melanoma-associated antigens, mutated p53, mutated ras, etc. In some embodiments, the CAR may be co-expressed with cytokines to improve persistence when low amounts of the tumor-associated antigen are present. For example, the CAR may be co-expressed with IL-15.

[0212] The sequence encoding the open reading frame of the chimeric receptor can be obtained from genomic DNA, cDNA, or can be synthetic (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, as introns are found to stabilize the mRNA. Furthermore, it may be even more advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0213] It is considered that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods for stably transfecting cells using naked DNA via electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA that encodes a chimeric receptor contained in a plasmid expression vector with appropriate orientation for expression.

[0214] Alternatively, a viral vector (e.g., a retroviral vector, adenovirus vector, adeno-associated virus vector, or lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors used in accordance with the methods of this disclosure are non-replicative in the immune cells. A large number of virus-based vectors are known in which the viral copy number in the cells is maintained sufficiently low to preserve the viability of the cells, such as vectors based on HIV, SV40, EBV, HSV, or BPV.

[0215] In some aspects, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to an extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated 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 prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0216] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., transmembrane regions containing at least the following molecules): the α, β, or ζ chain of a T-cell receptor, CD28, CD2, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8 (including CD8α), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, PD-1, CTLA4, and DAP molecules. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain primarily contains hydrophobic residues, such as leucine and valine. In some respects, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthesized transmembrane domain.

[0217] The hinge region of the CAR may be located at the N-terminus of the transmembrane domain and, in some embodiments, is derived from a natural or synthetic source. The hinge sequence may also be referred to as a spacer or extracellular spacer and is typically the extracellular structural region of the CAR that separates the binding unit from the transmembrane domain. In a particular embodiment, the CAR comprises an immunoglobulin (Ig)-like domain hinge. The hinge typically provides stability for efficient CAR expression and activity. The hinges may be from any suitable source, but in particular embodiments, several hinges are derived from CD8a, CD28, PD-1, CTLA4, the α, β, or ζ chain of the T-cell receptor, CD2, CD3ζ, CD3ε, CD3γ, CD3δ, 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 combinations thereof or others.

[0218] In some embodiments, the platform technologies disclosed herein for genetically modified immune cells (e.g., T or NK cells) include: (i) nonviral gene transfer using an electroporation device (e.g., a nucleofector); (ii) CARs that signal via intracellular domains (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations); (iii) CARs having extracellular domains of variable length that link antigen recognition domains to the cell surface; and in some cases, (iv) artificial antigen-presenting cells (aAPCs) derived from K562, enabling robust and digital amplification of CARs. + Immune cells (Singh et al., 2008; Singh et al., 2011).

[0219] In some embodiments, the cells are engineered to express a CD19-CAR sequence (SEQ ID NO:26) comprising 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 a transmembrane region, as well as CD3 and CD28 signal transduction regions.

[0220]

[0221] Specific examples of CARs that can be used (FMC63-CD8a hinge / TM-CD28-CD3z) are as follows:

[0222] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGG TKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:27).

[0223] FMC63-CD8a hinge / TM-CD28-CD3z

[0224] An example of an anti-CD19 CAR is shown below, which includes an anti-CD19scFv FMC63, a CD8a hinge and transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ (FMC63-CD8a hinge / TM-CD28-CD3z):

[0225]

[0226] In the example of SEQ ID NO:28, the following components of the CAR are described:

[0227] CD8 signal peptide

[0228] ATGGCCCTGCCAGTGACCGCCCTGCTGCTGCCACTGGCACTGCTGCTGCACGCAGCAAGGCCA(SEQID NO:29)

[0229] FMC63 Light Chain

[0230] GACATCCAGATGACAGACCACAAGCTCCCTGTCCGCCTCTCTGGGCGACAGAGTGACCATCTCTTGCAGGGCCAGCCAGGATATCTCCAAGTATCTGAATTGGTACCAGCAGAAGCCTGATGGCACAGTGAAGCTGCTGATCTATCACACCTCTAGACTG CACAGCGGCTGCCATCCAGGTTTAGCGGCTCCGGCTCTGGCACAGACTACTCTCTGACCATCAGCAATCTGGAGCAGGAGGATATCGCCACCTATTTCTGCCAGCAGGGCAACACACTGCCTTACACCTTTGGCGGCGGCACAAAAGCTGGAGATCACC(SEQ ID NO:30)

[0231] Connector

[0232] GGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCC (SEQ ID NO:31)

[0233] Heavy chain

[0234] GAGGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCACCCAGCCAGTCCCTGTCTGTGACATGTACCGTGTCCGGCGTGTCTCTGCCAGACTACGGCGTGAGCTGGATCAGACAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCCGAGACCACATACTATAACTCCGCCCTGAAGTCTCGGCTGACCATCATCAAGGACAACAGCAAGTCCCAGGTGTTTCTGAAGATGAATTCCCTGCAGACAGACGATACCGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCTTATGCCATGGATTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGC(SEQ ID NO:32)

[0235] CD8a hinge

[0236] ACCACAACCCCTGCACCAAGACCACCAACACCAGCACCTACCATCGCAAGCCAGCCTCTGTCCCTGAGGCCAGAGGCATGCAGGCCAGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTCGCCTGCGAT(SEQ IDNO:33)

[0237] CD8TM

[0238] ATCTACATCTGGGCACCACTGGCAGGAACATGTGGAGTGCTGCTGCTGTCTCTGGTCATCACCCTGTATTGTTGGGTG(SEQ ID NO:34)

[0239] CD28 co - stimulatory domain

[0240] AGAAGCAAGAGATCCAGGCTGCTGCACAGCGACTACATGAATATGACACCAAGGAGACCAGGACCAACCAGGAAGCACTATCAGCCTTACGCACCTCCAAGGGACTTCGCAGCATATAGGAGC(SEQ ID NO:35)

[0241] CD3ζ

[0242] AGGGTGAAGTTTTCTCGCAGCGCCGATGCCCCAGCCTATcAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGAGGAGAGGAAGGGATCCAGAGATGGGAGGCAAGCCTAGGCGCAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGAGAGAGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGTCCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCACTGCCACCTAGA(SEQ ID NO:36).

[0243] The corresponding amino acid sequence of FMC63-CD8a hinge / TM-CD28-CD3z is as follows:

[0244] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:37).

[0245] In the example of SEQ ID NO:37, the following components of the CAR are described:

[0246] CD8 signal peptide

[0247] MALPVTALLLPLALLLHAARP(SEQ ID NO:38)

[0248] FMC63 Light Chain

[0249]

[0250] Connector

[0251] GGGGSGGGGSGGGGS(SEQ ID NO:40)

[0252] Heavy chain

[0253]

[0254] CD8a hinge

[0255] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:42)

[0256] CD8TM

[0257] IYIWAPLAGTCGVLLLSLVITLYCWV(SEQ ID NO:43)

[0258] CD28 costimulatory domain

[0259] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:44)

[0260] CD3ζ

[0261] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 45).

[0262] FMC63-CD28 hinge / TM-CD28-CD3z

[0263] An example of an anti-CD19 CAR is shown below, which includes an anti-CD19scFv FMC63, a CD28 hinge and transmembrane domain, a CD28 co-stimulatory domain, and CD3ζ (FMC63-CD28 hinge / TM-CD28-CD3z):

[0264]

[0265] The amino acid sequence of FMC63-CD28 hinge / TM-CD28-CD3z is as follows:

[0266] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:47).

[0267] Another example of the nucleic acid sequence of FMC63-CD28 hinge-TM CAR is as follows:

[0268]

[0269] CD28 hinge:

[0270] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 49).

[0271] CD28 hinge nucleic acid sequence:

[0272] ATCGAAGTGATGTATCCACCCCCTTACCTGGATAACGAGAAGAGCAATGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCATCTCCCCTGTTCCCTGGCCCAAGCAAGCCC (SEQ ID NO: 50).

[0273] CD28 TM structure domain:

[0274] FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 51).

[0275] FMC63-PD-1 Hinge-TM CAR

[0276] An example of a CAR having the following components is: CSF2RA signal peptide-FMC63 light chain-linker-heavy chain-PD1 hinge-PD-1TM-CD28 Costim-CD3ζ, as follows:

[0277] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSQVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVIERSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:52).

[0278] The nucleic acid sequence of FMC63-PD-1 hinge-TM CAR is as follows:

[0279]

[0280] PD-1 hinge

[0281] QVPTAHPSPSPRPAGQFQTLV (SEQ ID NO:54).

[0282] PD-1 TM domain

[0283] VGVVGGLLGSLVLLVWVLAVI (SEQ ID NO:55).

[0284] FMC63-CTLA4 hinge -™ CAR:

[0285] CSF2RA signal peptide - FMC63 light chain - linker - heavy chain - CTLA4 hinge – CTLA-4 TM - CD28 Cost - CD3ζ

[0286] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSvidpepcpdsdfllwilaavssglffysflltaRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:56)

[0287]

[0288] CSF2RA signal peptide

[0289] MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO:58).

[0290] CTLA4 hinge

[0291] VIDPEPCPDSD(SEQ ID NO:59).

[0292] CTLA4™ domain

[0293] FLLWILAAVSSGLFFYSFLLT (SEQ ID NO: 60).

[0294] BT cell receptor (TCR)

[0295] In some embodiments, the genetically modified antigen receptor comprises a recombinant TCR and / or a TCR cloned from naturally occurring T cells. A “T cell receptor” or “TCR” refers to a molecule containing variable α and β chains (also referred to as TCRα and TCRβ, respectively) or variable γ and δ chains (also referred to as TCRγ and TCRδ, respectively) and capable of specifically binding to an antigen that binds to an MHC receptor. In some embodiments, the TCR is in αβ form. In alternative embodiments, the cells lack a modified TCR; for example, an endogenous TCR in the cells can target cancer or infectious diseases (e.g., CMV or EBV-specific T cells with an endogenous TCR).

[0296] Typically, TCRs existing in αβ and γδ forms are structurally similar, but T cells expressing them can have different anatomical locations or functions. TCRs can be found on the cell surface or in a soluble form. Typically, TCRs are found on the surface of T cells (or T lymphocytes), where they are usually responsible for recognizing antigens that bind to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, for example, Janeway et al., 1997). For example, in some aspects, 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 are associated with the invariant protein of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term “TCR” should be understood to encompass its functional TCR fragments. The term also encompasses complete or full-length TCRs, including TCRs in αβ or γδ form.

[0297] Therefore, for the purposes of this document, reference to TCR includes any TCR or functional fragment, such as the antigen-binding moiety of a TCR, that binds to a specific antigenic peptide (i.e., an MHC-peptide complex) bound to an MHC molecule. The term "antigen-binding moiety" or "antigen-binding fragment" of a TCR (these terms are used interchangeably) refers to a molecule that contains a portion of the structural domains of a TCR but binds to the antigen (e.g., an MHC-peptide complex) that the full TCR binds to. In some cases, the antigen-binding moiety contains variable domains of the TCR, such as variable α-chains and variable β-chains of the TCR, sufficient to form binding sites for binding to a specific MHC-peptide complex, typically where each chain contains three complementarity-determining regions.

[0298] In some embodiments, the variable domains of the TCR chain associate to form loops or complementarity-determining regions (CDRs) similar to those of immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, for example, Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the major CDR responsible for recognizing the processed antigen, although CDR1 of the α chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β chain may include a further highly variable (HV4) region.

[0299] In some embodiments, the TCR chain comprises constant domains. For example, like immunoglobulins, the extracellular portion of the TCR chain (e.g., α-chain, β-chain) may contain two immunoglobulin domains and a variable domain at the N-terminus (e.g., V...). a Or Vp; typically, based on amino acid 1 to 116 numbered by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," US Dept. Health and Human Services, Public Health Service, National Institutes of Health, 1991, 5th edition), and a constant domain adjacent to the cell membrane (e.g., α-chain constant domain or C). aTypically, the β-chain constant domain or Cp is based on Kabat amino acids 117 to 259; typically, it is based on Kabat amino acids 117 to 295. For example, in some cases, the extracellular portion of the TCR formed by the two chains comprises two proximal membrane constant domains and two distal membrane variable domains containing CDRs. The constant domains of the TCR contain short linker sequences in which cysteine ​​residues form disulfide bonds, thereby constituting the link between the two chains. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α and β chains, so that the TCR contains two disulfide bonds in the constant domain.

[0300] 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 region. In some cases, the structure allows the TCR to associate with other molecules such as CD3. For example, a TCR containing a constant domain with a transmembrane region can anchor to proteins in the cell membrane and associate with invariant subunits of CD3 signaling transducers or complexes.

[0301] Typically, CD3 is a multi-protein complex that can have three distinct chains (γ, δ, and ε) (in mammals) and a ζ-chain. For example, in mammals, the complex can consist of a homodimer of one CD3γ chain, one CD3δ chain, two CD3ε chains, and a CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly associated cell surface proteins of the immunoglobulin superfamily, each containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature 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 (called an immunoreceptor tyrosine-based activation motif, or ITAM), while each CD3ζ chain has three. Typically, ITAMs are involved in the signal transduction capabilities of the TCR complex. These associated molecules have negatively charged transmembrane regions and play a role in transmitting signals from the TCR into the cell. The CD3- and ζ- chains together with the TCR form the so-called T-cell receptor complex.

[0302] In some embodiments, the TCR may be a heterodimer of two strands, α and β (or optionally γ and δ), or it may be a single-stranded TCR construct. In some embodiments, the TCR is a heterodimer comprising two linked (e.g., by disulfide bonds) separate strands (α and β chains or γ and δ chains). In some embodiments, a TCR is identified with respect to a target antigen (e.g., a cancer antigen) and introduced into cells. In some embodiments, the nucleic acid polymer encoding the TCR may be obtained from a wide variety of sources, such as by polymerase chain reaction (PCR) amplification of a publicly available TCR DNA sequence. In some embodiments, the TCR is obtained from a biological source, such as cells like T cells (e.g., cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, the T cells may be cells isolated from within the body. In some embodiments, high-affinity T cell clones may be isolated from a patient, and the TCR may be isolated. In some embodiments, the T cells may be cultured T cell hybridomas or clones. In some embodiments, a TCR clone targeting the target antigen is generated in transgenic mice modified with human immune system genes (e.g., the human leukocyte antigen system or HLA). See, for example, tumor antigens (see, for example, Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate the TCR targeting the target antigen (see, for example, Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or its antigen-binding portion can be generated synthetically from knowledge of the TCR's sequence.

[0303] C. Antigen-presenting cells

[0304] 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 a portion of the antigen along with the MHC molecule on their outer cell membrane. The MHC is a large genetic complex with multiple loci. These MHC loci encode two main classes of MHC membrane molecules, known as class I and class II MHCs. T helper lymphocytes typically recognize antigens associated with class II MHC molecules, while T cytotoxic lymphocytes recognize antigens associated with class I MHC molecules. In humans, the MHC is referred to as the HLA complex, and in mice as the H-2 complex.

[0305] In some cases, APCs are useful in the preparation of therapeutic compositions and cell therapy products of the present embodiments. For general guidance on the preparation and use of antigen presentation systems, see, for example, U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662; U.S. Patent Application Publications Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. WO2007 / 103009.

[0306] An APC system may contain at least one exogenous helper molecule. Any suitable number and combination of helper molecules may be used. The helper molecules may be selected from helper molecules such as co-stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD86, CD64 (FcγRI), 41BB ligand, and IL-21. Adhesion molecules may include carbohydrate-binding glycoproteins such as select proteins, transmembrane-binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), which promote, for example, cell-cell or cell-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, and reagents useful for selecting, cloning, preparing, and expressing exemplary helper molecules (including co-stimulatory molecules and adhesion molecules) are exemplified, for example, in U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

[0307] D. Antigen

[0308] Among antigens targeted by genetically modified antigen receptors or naturally expressed antigen receptors (e.g., TCRs) on an unlimited number of immune cells are those expressed in the context of a disease, condition, or cell type to be targeted via adoptive cell therapy. These diseases and conditions include proliferative, neoplastic, and malignant diseases and conditions, including cancers and tumors, including hematologic cancers, cancers of the immune system such as lymphoma, leukemia, and / or myeloma, such as B, T, and myeloid leukemias, lymphoma, and multiple myeloma. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition (e.g., tumors or pathogenic cells) compared to normal or untargeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on modified cells.

[0309] Any suitable antigen can be used in the methods of this invention. Exemplary antigens include, but are not limited to, antigenic molecules derived from infectious agents, self / autoantigens, tumor / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In particular, said 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, and CEA. In particular, antigens for one or more of said 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. The sequences of 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 ... EGFRv1 (accession number NG_029383.1), HER2 (accession number NG_007275.1), EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), WT123 (accession number NC_00002 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).

[0310] Tumor-associated antigens can be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, or melanoma. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE 1, MAGE 3, and MAGE 4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); 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. See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostate acid phosphatase, NKX3.1, and prostate six-transmembrane epithelial antigen (STEAP).

[0311] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, tumor antigens may be autopeptide hormones, such as full-length gonadotropin-releasing hormone (GnRH) (a short peptide of 10 amino acids), which is useful in the treatment of many cancers.

[0312] Tumor antigens include those derived from cancers characterized by the expression of tumor-associated antigens (e.g., HER-2 / neu expression). Target tumor-associated antigens include lineage-specific tumor antigens, such as melanocyte-melanoma lineage antigens MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinases, and tyrosinase-related proteins. Illustrative tumor-associated antigens include, but are not limited to, tumor antigens derived from or containing one or more of the following: 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, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, ... GAGE-4, GAGE-5, GAGE-6, GAGE-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, phosphoinositol 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wirmes tumor antigen (WT1), AFP, β-linkin / m, caspase-8 / m, C EA, 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 Transducer 1 (TACSTD1), TACSTD2, and other related substances. Somatic tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-coupled kinases (ILK), signal transduction proteins and transcription activators STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-κB (NF-κB), Notch receptors (e.g., Notch1-4), c-Met,mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and its regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-α, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2ETS fusion) (Synthetic genes), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucose GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLOBOH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, spermin 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-associated antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and idiotypes.

[0313] Antigens may include epitope regions or epitope peptides derived from genes mutated in tumor cells or genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase, survivin, mesothelin, mutant ras, bcr / abl rearrangement, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intron sequences such as N-acetylglucosamine transferase-V; clonal rearrangements of immunoglobulin genes that generate unique types in myeloma and B-cell lymphoma; tumor antigens containing epitope regions or epitope peptides derived from tumor viral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and non-mutant carcinoembryonic proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0314] In some embodiments, the antigen may be microbial. In some embodiments, the antigen is obtained from or derived from pathogenic or opportunistic pathogenic microorganisms (also referred to herein as infectious disease microorganisms), such as viruses, fungi, parasites, and bacteria. In some embodiments, antigens derived from such microorganisms comprise full-length proteins.

[0315] The antigens considered for use in the methods described herein include illustrative pathogenic organisms such as 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 and JC viruses), adenoviruses, 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 those skilled in the art will understand, the proteins derived from these and other pathogenic microorganisms used as antigens described herein, as well as the nucleotide sequences encoding said proteins, can be found in publications and in public databases such as... SWISS- and It was identified in the middle.

[0316] Antigens derived from human immunodeficiency virus (HIV) include any of the following: HIV viral particle structural proteins (e.g., gp120, gp41, p17, p24), proteases, reverse transcriptases, or HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0317] Antigens derived from herpes simplex virus (e.g., HSV 1 and HSV 2) include, but are not limited to, proteins expressed from late-stage HSV genes. The late-stage genome primarily encodes proteins that form viral particles. These proteins include five proteins (ULs) that form the viral capsid: UL6, UL18, UL35, UL38, and the major capsid proteins UL19, UL45, and UL27, each of which can be used as antigens as described herein. Other illustrative HSV proteins considered for use as antigens herein include ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD). The HSV genome comprises at least 74 genes, each encoding a protein that can potentially be used as an antigen.

[0318] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed in the immediate early and early stages of viral replication, glycoproteins I and III, capsid proteins, outer coat proteins, submatrix proteins pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products from the gene cluster UL128-UL150 (Rykman et al., 2006), envelope glycoprotein B (gB), gH, gN, and pp150. As those skilled in the art will understand, the CMV proteins used as antigens described herein can be found in public databases such as […]. SWISS- and It was identified in (see, for example, Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[0319] Antigens derived from Epstein-Barr virus (EBV) that are considered for use in some embodiments include: EBV cleavage proteins gp350 and gp110, and EBV proteins produced during the latent period of infection, including EB 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, for example, Lockey et al., 2008).

[0320] The antigens derived from respiratory syncytial virus (RSV) considered for use in this paper include any one of the eleven proteins or antigenic fragments 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 regulator), RNA polymerase, and phosphoprotein P.

[0321] Antigens derived from vesicular stomatitis virus (VSV) considered for use include any one of the five major proteins encoded by the VSV genome and their antigenic fragments: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, for example, Rieder et al., 1999).

[0322] Antigens derived from influenza viruses that are considered for use in some implementation schemes include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[0323] Exemplary viral antigens also include, but are not limited to, adenovirus peptides, alphavirus peptides, calicivirus peptides (e.g., calicivirus capsid antigen), coronavirus peptides, warm virus peptides, Ebola virus peptides, enterovirus peptides, flavivirus peptides, hepatitis virus (AE) peptides (hepatitis B core or surface antigen, hepatitis C virus E1 or E2 glycoprotein, core or non-structural protein), herpesvirus peptides (including herpes simplex virus or varicella-zoster virus glycoprotein), infectious peritonitis virus peptides, leukemia virus peptides, Marburg virus peptides, orthomyxovirus peptides, papillomavirus peptides, parainfluenza virus peptides (e.g., hemagglutinin and neuraminidase peptides), paramyxovirus peptides, parvovirus peptides, plague virus peptides, small RNA virus peptides (e.g., poliovirus capsid peptides), poxvirus peptides (e.g., vaccinia virus peptides), rabies virus peptides (e.g., rabies virus glycoprotein G), reovirus peptides, retrovirus peptides, and rotavirus peptides.

[0324] In some embodiments, the antigen may be a bacterial antigen. In some embodiments, the target bacterial antigen may be a secreted polypeptide. In other embodiments, the bacterial antigen comprises an antigen having a portion of a polypeptide exposed on the outer cell surface of the bacteria.

[0325] Antigens derived from Staphylococcus species (including methicillin-resistant Staphylococcus aureus (MRSA)) considered for use include: virulence modifiers such as the Agr system, Sar and Sae, the Arl system, Sar homologs (Rot, McGrath A, SarS, SarR, SarT, SarU, SarV, SarX, SarZ, and TcaR), the Srr system, and TRAP. Other Staphylococcus proteins that can serve as antigens include Clp protein, HtrA, MsrR, cis-aconitase, CcpA, SvrA, Msa, CfvA, and CfvB (see, for example, Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, edited by Jodi Lindsay). The genomes of two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example in PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As those skilled in the art will understand, Staphylococcus proteins used as antigens are also available in other public databases, such as... Swiss- and It was identified in the middle.

[0326] Antigens derived from *Streptococcus pneumoniae* that are considered for use in some of the embodiments described herein include: pneumococcal hemolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and fimbriae proteins (RrgA; RrgB; RrgC). Antigenic proteins of *Streptococcus pneumoniae* are also known in the art and can be used as antigens in some embodiments (see, for example, Zysk et al., 2000). The complete genome sequences of virulent strains of *Streptococcus pneumoniae* have been sequenced, and as those skilled in the art will understand, the *Streptococcus pneumoniae* strains used herein can also be found in other public databases, such as... and The proteins identified are, for example, virulence factors and proteins predicted to be exposed on the surface of pneumococci (see, for example, Frolet et al., 2010).

[0327] Examples of bacterial antigens that can be used as antigens include, but are not limited to: Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, and Chlamydia polypeptides. Mydia polypeptide, Corynebacterium polypeptide, Coxiella polypeptide, Dermatophilus polypeptide, Enterococcus polypeptide, Ehrlichia polypeptide, Escherichia polypeptide, Francisella polypeptide, Fusobacterium polypeptide, Haemobartonella polypeptide, Haemophilus polypeptide (e.g., Haemophilus influenzae) polypeptide, E. influenzae (e.g., Haemophilus influenzae) polypeptide, E. influenzae (e.g., Haemophilus influenzae) polypeptide, E. influenzae (e.g., E. ... e) Type b outer membrane protein), Helicobacter polypeptide, Klebsiella polypeptide, L-type bacterial polypeptide, Leptospira polypeptide, Listeria polypeptide, Mycobacteria polypeptide, Mycoplasma polypeptide, Neisseria polypeptide, Neorikettsia polypeptide, Nocardia polypeptide, Pasteurella polypeptide, Peptococcus polypeptide Peptostreptococcus polypeptide, Pneumococcus polypeptide (i.e., S. pneumoniae polypeptide) (see description herein), Proteus polypeptide, Pseudomonas polypeptide, Rickettsia polypeptide, Rochalimaea polypeptide, Salmonella polypeptide, Shigella polypeptide, Staphylococcus polypeptide, Group A streptococcus polypeptide (e.g., Streptococcus pyogenes).(e.g., Yersinia pestis F1 and V antigens) (such as M protein from *Streptococcus pyogenes*, peptides from Group B streptococci (*Streptococcus agalactiae*), peptides from *Treponema*, and peptides from *Yersinia pestis*).

[0328] Examples of fungal antigens include, but are not limited to: *Absidia* polypeptide, *Acremonium* polypeptide, *Alternaria* polypeptide, *Aspergillus* polypeptide, *Basidiobolus* polypeptide, *Bipolaris* polypeptide, *Blastomyces* polypeptide, *Candida* polypeptide, *Coccidioides* polypeptide, *Conidiobolus* polypeptide, *Cryptococcus* polypeptide, *Curvalaria* polypeptide, *Epidermophyton* polypeptide, *Exophiala* polypeptide, *Geotrichum* polypeptide, *Histoplasma* polypeptide, *Madurella* polypeptide, *Malassezia* polypeptide, and *Microsporum* polypeptide. Polypeptides from the genera *Moniliella*, *Mortierella*, *Mucor*, *Paecilomyces*, *Penicillium*, *Phialemonium*, *Phialophora*, *Prototheca*, *Pseudallescheria*, and *Pseudomi*. The following peptides are listed: crodochium peptide, Pythium peptide, Rhinosporidium peptide, Rhizopus peptide, Scolecobasidium peptide, Sporothrix peptide, Stemphylium peptide, Trichophyton peptide, Trichosporon peptide, and Xylohypha peptide.

[0329] 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 worm parasite antigens include, but are not limited to: peptides from the genera *Acanthocheilonema*, *Aelurostrongylus*, *Ancylostoma*, *Angiostrongylus*, *Ascaris*, *Brugia*, *Bunostomum*, *Capillaria*, *Chabertia*, *Cooperia*, *Crenosoma*, *Dictyocaulus*, *Dioctophyme*, *Dipetalonema*, and *Dip*. Polypeptides from the genera *Hylobothrium*, *Dipylidium*, *Dirofilaria*, *Dracunculus*, *Enterobius*, *Filaroides*, *Haemonchus*, *Lagochilascaris*, *Loa*, *Mansonella*, *Muellerius*, *Nanophyetus*, *Necator*, *Nematodirus*, *Oesophagostomum*, and *Onchocerca*.Polypeptides from the genera *Opisthorchis*, *Ostertagia*, *Parafilaria*, *Paragonimus*, *Parascaris*, *Physaloptera*, *Protostrongylus*, *Setaria*, *Spirocerca*, *Spirometra*, and *Ste...* The following peptides are listed: phanofilaria peptide, Strongyloides peptide, Strongylus peptide, Thelazia peptide, Toxascaris peptide, Toxocara peptide, Trichinella peptide, Trichostrongylus peptide, Trichuris peptide, Uncinaria peptide, and Wuchereria peptide. (For example, cyclospores of Plasmodium falciparum (PfCSP), sporospore surface protein 2 (PfSSP2), the carboxyl terminus of hepatic stage antigen 1 (PfLSA1c-term), and export protein 1 (PfExp-1)); peptides from the genera Pneumocystis, Sarcocystis, Schistosoma, Theileria, Toxoplasma, and Trypanosoma.)

[0330] Examples of ectoparasites include, but are not limited to, polypeptides (including antigens and allergens) from the following: fleas; ticks, including hard ticks and soft ticks; flies, such as midges, mosquitoes, sandflies, black flies, horseflies, hornflies, tussock flies, tsetse flies, stable flies, flies that cause myiasis and biting flies; ants; spiders; lice; mites; and stink bugs, such as bedbugs and assassin bugs.

[0331] E. Suicide gene

[0332] The unlimited immune cells of this disclosure (including those capable of expressing one or more CARs and / or one or more modified TCRs) may contain one or more suicide genes. As used herein, the term "suicide gene" is defined as a gene that, upon administration of a prodrug, converts its gene product into a compound that kills its host cells. Examples of possible suicide gene / prodrug combinations are 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 cytarabine.

[0333] V. Methods of delivery to cells

[0334] Those skilled in the art will be able to construct vectors for the expression of antigen receptors of this disclosure using standard recombination techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference). Vectors include, but are not limited to: plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses) and artificial chromosomes (e.g., YAC), such as retroviral vectors (e.g., derived from Moloney mouse leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors (including their replicative, replication-defective, and content-free forms), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey mouse sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, maraba virus vectors, and group B adenovirus enadenotucirev vectors.

[0335] A. Viral vector

[0336] In certain aspects of this disclosure, viral vectors encoding BCL6 and cell survival-promoting genes and / or antigen receptors may be provided. In the generation of recombinant viral vectors, non-essential genes are typically replaced by genes or coding sequences concerning heterologous (or non-natural) proteins. Viral vectors are a class of expression constructs that utilize viral sequences to introduce nucleic acid polymers and, possibly, proteins, into cells. The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis and integrate into the host cell genome and to stably and efficiently express viral genes makes them attractive candidates for the transfer of 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 of certain aspects of this disclosure are described below.

[0337] Lentivirals are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, for example, U.S. Patents 6,013,516 and 5,994,136).

[0338] Recombinant lentiviruses can infect non-dividing cells and can be used for in vivo and in vitro gene transfer and expression of nucleic acid polymer sequences. For example, recombinant lentiviruses capable of infecting non-dividing cells—in which suitable host cells are transfected with two or more vectors carrying packaging functions, namely gag, pol, and env, and rev and tat—are described in U.S. Patent 5,994,136 (which is incorporated herein by reference).

[0339] B. Control elements

[0340] The expression cassettes contained in vectors useful in this disclosure specifically include (in a 5' to -3' orientation) eukaryotic transcription promoters, splicing signals (including intercalation sequences), and transcription termination / polyadenylation sequences operatively linked to protein-coding sequences. In eukaryotic cells, promoters and enhancers controlling transcription of protein-coding genes consist of multiple genetic elements. Cellular machinery can collect and integrate the regulatory information conveyed by each element, thereby allowing different genes to develop different, often complex, transcriptional regulatory patterns. Promoters used in the context of this disclosure include constitutive, inducible, and tissue-specific promoters.

[0341] C. Promoters / Enhancers

[0342] The expression constructs presented herein contain promoters to drive the expression of antigen receptors. Promoters typically contain sequences that function to place the initiation site for RNA synthesis. The most well-known example of this is the TATA box, but in some promoters lacking the TATA box, such as those for mammalian terminal deoxynucleotidyl transferase genes and SV40 late-stage genes, discrete elements superimposed on the initiation site itself help to fix the initiation position. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in a region 30-110 bp upstream of the initiation site, although many promoters have been shown to also contain functional elements downstream of the initiation site. To bring the coding sequence under the “control” of the promoter, the 5′ end of the transcription start site of the transcription reading frame is placed “downstream” (i.e., 3′) of the selected promoter. The “upstream” promoter stimulates transcription of the DNA and promotes the expression of the encoded RNA.

[0343] The spacing between promoter elements is often flexible, thus preserving promoter function when an element is inverted or moved relative to another element. In the tk promoter, the spacing between promoter elements can increase to 50 bp, after which activity begins to decline. Depending on the promoter, it appears that individual elements can function both cooperatively and independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which are cis-regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0344] Promoters can be promoters naturally associated with a nucleic acid sequence, such as those obtained by isolating a 5′-noncoding sequence located upstream of a coding region and / or exon. Such promoters can be called “endogenous.” Similarly, enhancers can be enhancers naturally associated with a nucleic acid sequence, located downstream or upstream of that sequence. Alternatively, certain advantages may be gained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter (which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment). Recombinant or heterologous enhancers are also enhancers that are not normally associated with a nucleic acid sequence in their natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are not “naturally occurring,” i.e., those containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, the promoters most frequently used in recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to generating promoter and enhancer nucleic acid sequences synthetically, the compositions disclosed herein can also be used by recombinant cloning and / or nucleic acid amplification techniques (including PCR). TMSequences can be generated using [a specific method / mechanism]. Furthermore, it has been considered that control sequences could also be used to guide the transcription and / or expression of sequences within non-nuclear organelles (e.g., mitochondria, chloroplasts, etc.).

[0345] Naturally, it will be important to employ promoters and / or enhancers that effectively direct the expression of DNA segments in the organelles, cell types, tissues, organs, or organisms selected for expression. Those skilled in the art of molecular biology are generally familiar with the use of combinations of promoters, enhancers, and cell types for protein expression (see, for example, Sambrook et al., 1989, which are incorporated herein by reference). The promoters employed can be constitutive, tissue-specific, inducible, and / or, under suitable conditions, useful for directing high-level expression of the introduced DNA segment, for example, advantageous in the large-scale production of recombinant proteins and / or peptides. The promoters can be heterologous or endogenous.

[0346] Alternatively, expression can be driven using any promoter / enhancer combination (according to, for example, the eukaryotic promoter database EPDB, available at epd.isb-sib.ch / via the World Wide Web). The use of T3, T7, or SP6 cytoplasmic expression systems is another possible implementation. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters, provided a suitable bacterial polymerase is available, either as part of a delivery complex or as an additional genetic expression construct.

[0347] Non-limiting examples of promoters include: early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus (RSV) early promoter; eukaryotic cell promoters, such as the β-actin promoter, the GADPH promoter, and the metallothionein promoter; and cascaded response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the response element promoter (tre) near the minimal TATA box. It is also possible to use human growth hormone promoter sequences (e.g., the minimal human growth hormone promoter described in Genbank, accession number X05244, nucleotides 283-341) or mouse mammary tumor promoters (available from ATCC, catalog number ATCC 45007). In some embodiments, the promoter is CMV IE, dectin-1, dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, β-actin, a class I MHC or class II MHC promoter, but any other promoter useful for driving the expression of therapeutic genes may also be applicable to the practice of this disclosure.

[0348] In some respects, the methods of this disclosure also involve enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and have the potential to function cis- and regardless of orientation, even at relatively long distances (up to several thousand bases away from the target promoter). However, enhancer function is not necessarily limited to such long distances, as they can also function very close to a given promoter.

[0349] D. Initial signals and linkage expression

[0350] Specific start signals may also be used in the expression constructs provided in this disclosure for efficient translation of the coding sequence. These signals include the ATG start codon or a neighboring sequence. Exogenous translation control signals, including the ATG start codon, may need to be provided. Those skilled in the art will readily be able to determine this and provide the necessary signals. It is well known that the start codon must "frame-match" with the desired coding sequence's reading frame to ensure translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic. Expression efficiency can be enhanced by including suitable transcriptional enhancer elements.

[0351] In some implementations, internal ribosome entry site (IRES) elements are used to create multi-gene or polycistronic information. IRES elements bypass the cap-dependent ribosome scanning pattern of 5' methylation translation and initiate translation at an internal site. IRES elements from two members of the small RNA virus family (poliovirus and encephalomyovirus), as well as IRES from mammalian information, have been described. IRES elements can be linked to heterologous open reading frames (OPFs). Multiple OPFs can be transcribed together, each separated by an IRES, thus producing polycistronic information. With IRES elements, each OPF is accessible to the ribosome for efficient translation. Multiple genes can be efficiently expressed by transcribing a single message using a single promoter / enhancer.

[0352] Additionally, certain 2A sequence elements can be used to generate synergistic or co-expression of genes within the constructs provided in this disclosure. For example, the cleavage sequence can be used to co-express genes by connecting open reading frames to form a single cistron. An exemplary cleavage sequence is F2A (foot-and-mouth disease virus 2A) or a "2A-like" sequence (e.g., Thosea asigna virus 2A; T2A).

[0353] E. Replication Origin

[0354] To propagate the vector in host cells, it may contain one or more origin of replication sites (commonly referred to as "ori"), for example, a nucleic acid sequence corresponding to the oriP of EBV described above, or a genetically modified oriP with similar or enhanced programmed function, which is the specific nucleic acid sequence at which replication begins. Alternatively, origin of replication or autonomous replication sequence (ARS) of other extrachromosomal replicating viruses described above may be used.

[0355] F. Selecting and Filtering Markers

[0356] In some embodiments, cells containing constructs of this disclosure can be identified in vitro or in vivo by including markers in the expression vector. Such markers confer identifiable changes to the cells, thereby allowing easy identification of cells containing the expression vector. Typically, selection markers are markers that impart properties that allow selection. Positive selection markers are those whose presence allows selection, while negative selection markers are those whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0357] Typically, drug selection markers are helpful for the cloning and identification of transformants; for example, conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histamine are useful selection markers. In addition to markers that allow for condition-based differentiation of transformant phenotypes, other types of markers are considered, including screenable markers such as GFP, based on colorimetric analysis. Alternatively, screenable enzymes, such as herpes simplex virus thymidine kinase (TK) or chloramphenicol acetyltransferase (CAT), can be used as negative selection markers. Those skilled in the art will also know how to employ immunomarkers, possibly in conjunction with FACS analysis. The marker used is not considered important as long as it can be co-expressed with the nucleic acid encoding the gene product. Further examples of selection and screenable markers are well known to those skilled in the art.

[0358] G. Methods for delivering nucleic acid polymers

[0359] The modified immune cells can be constructed using many well-established gene transfer methods known to those skilled in the art. In some embodiments, the modified cells are constructed by introducing nucleic acid polymers using a viral vector-based gene transfer method. The viral vector-based gene transfer method may include lentiviral vectors, retroviral vectors, adenoviruses, or adeno-associated virus vectors. In some embodiments, the modified cells are constructed by introducing nucleic acid polymers using a non-viral vector-based gene transfer method. In some embodiments, the non-viral vector-based gene transfer method includes 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 some embodiments, the gene editing method based on a non-viral vector includes transfection or transformation methods selected from the group consisting of: lipid transfection, nucleofection, virion, liposome, polycationic or lipid:nucleoprotein conjugate, naked DNA, artificial viral particles, and reagent-enhanced uptake of DNA.

[0360] The cells can be modified to express target genes and / or antigen receptors through random or site-directed insertion, for example, through gene editing methods, including but not limited to megabase-wide nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system.

[0361] In addition to viral delivery of nucleic acid polymers encoding target genes and / or antigen receptors, the following are other methods for delivering recombinant genes to a given host cell and are therefore considered in this disclosure. The introduction of nucleic acid polymers, such as DNA or RNA, into the immune cells of this disclosure can be performed using any suitable method for nucleic acid polymer delivery to transform cells, which is described herein or will be known to those skilled in the art. Such methods include, but are not limited to: direct delivery of DNA, e.g., by in vitro transfection, by injection (including microinjection); by electroporation; by calcium phosphate precipitation; by the use of DEAE-glucan followed by polyethylene glycol; by direct acoustic loading; by liposome-mediated transfection and receptor-mediated transfection; by microparticle bombardment; by shaking with silicon carbide fibers; by Agrobacterium-mediated transformation; by drying / inhibition-mediated DNA uptake; and any combination of such methods. By applying techniques such as these, organelles, cells, tissues, or organisms can be transformed stably or transiently.

[0362] VI. Treatment methods

[0363] The unlimited immune cells of this invention can be used for both therapeutic and research purposes. The unlimited immune cells of this invention (including T cells or NK cells expressing CAR and / or modified TCRs) can be used to treat cancer, infectious diseases, immune disorders, or inflammatory conditions.

[0364] In one approach, allogeneic, off-the-shelf CAR T cells targeting antigens (e.g., CD19, CD20, CD22, CD79a, CD79b, or BAFF-R) can be used (alone or in combination) to treat B-cell leukemia and lymphoma. Allogeneic, off-the-shelf anti-mesothelin CAR T cells can be used to treat, for example, mesothelioma, pancreatic adenocarcinoma, or ovarian cancer. NY-ESO-targeting TCR-T cells can be used to treat, for example, melanoma or multiple myeloma. Virus-specific T cells targeting viruses such as EBV, CMV, BK virus, etc., can be used to treat their respective viral infections. Allogeneic suppressor or regulatory T cells can be used to treat autoimmune diseases, GVHD, and other inflammatory conditions.

[0365] In specific embodiments, γ / δ T cells and virus-specific T cells are unlikely to induce GvHD but provide additional antitumor and / or antiviral functions. In specific embodiments, virus-specific unlimited T cells can be used for at least two purposes. First, virus-specific unlimited T cells can be used to treat specific viral infections, such as CMV or EBV infection, or certain cancers. A second embodiment involves transducing one or more CARs and / or modified TCRs into virus-specific T cells. Such unlimited CAR T cells with virus-specific endogenous TCRs may have the potential advantage of being unlikely to induce GvHD. Such cells carrying virus-specific endogenous TCRs do not require gene editing methods to knock out the TCRs in said T cells. If gene editing technologies such as CRISPR / Cas9 are combined, then virus-specific T cells are not necessarily required to generate CAR-T cells. Alternatively, γ / δ unlimited CAR T cells, or CAR-NK or CAR-NKT or CAR-innate lymphoid cells, can be used; these do not induce GvHD and are not expected to require TCR knockout.

[0366] When intended for human use, the modified cell lines of the invention are first tested in animal models (such as the NSG mouse model, which is frequently used in cancer research) to assess tumor-killing activity and therapeutic efficacy. Such studies in mice are preclinical studies, which can be conducted prior to therapeutic use in patients.

[0367] The unlimited immune cells can be used to treat cancers, including hematologic and non-hematologic malignancies, for example by administering to a patient effective amounts of modified cytotoxic unlimited T cells (alone or in combination) expressing different CARs or TCRs targeting different tumor targets. For example, CD19 inCAR (one of which is Ie1-L4aJ3 cells (CD8-positive cells from healthy donor 1 transduced with a CAR targeting human CD19 with a truncated human EFGR marker)) can be administered together with IL-2 or IL-15 to treat patients with B-cell leukemia or lymphoma. The Ie1-L4aJ3 cells can be present in conventional pharmaceutical excipients (e.g., water or buffered saline). After administration to a patient, the modified cells can inhibit tumor growth through CD19-guided killing. For human patients, the immune cells can be administered via intravenous infusion (iv). However, other administration methods, such as subcutaneous (sc) injection, can be used. After successfully eradicating the neoplasm, the immune cells can be cleared by withdrawing IL-2 or IL-15 or by infusing anti-EGFR antibodies.

[0368] The appropriate dose of the unlimited immune cells (and one or more cytokines, such as IL-2 and / or IL-15, when used) depends on the recipient's age, health, sex, and weight, as well as any other concurrent treatments the recipient is undergoing for a related or unrelated condition. Those skilled in the art can readily determine the appropriate dose of modified cells and drugs to be administered to the patient, depending on the factors mentioned above. The number of cells constituting an effective tumor-killing dose can be determined using animal models. These parameters can be readily determined by those skilled in the art.

[0369] The effectiveness of this therapy against tumors can be determined by detecting any surviving tumor cells in a sample of the patient's peripheral blood or bone marrow, or by other diagnostic imaging studies such as CT, MRI, or PET scans. Similarly, any residual, unwanted, uncontrolled modified T cells can be monitored using methods such as flow cytometry and polymerase chain reaction.

[0370] Compared to previous cytotoxic cell lines such as TALL-104 and NK-92, unlimited immune cells are generated from normal immune cells. Therefore, the risk of leukemia induced by unlimited immune cells is lower than that of TALL-104 and NK-92, as unlimited immune cells are not expected to carry any other unknown tumorigenic genetic mutations. Furthermore, the proliferation of unlimited cells can be stopped by interrupting IL-2 or IL-15. This is an unparalleled safety advantage compared to the leukemia-derived cell lines TALL-104 and NK-92.

[0371] In some embodiments, this disclosure provides methods for immunotherapy, comprising administering an effective amount of immune cells of this disclosure. In some embodiments of this disclosure, cancer or infection is treated by transferring a population of immune cells that elicit an immune response. Methods for treating cancer or delaying cancer progression in an individual are provided herein, comprising administering an effective amount of antigen-specific cell therapy to said individual. This method can be applied to the treatment of immune disorders, solid tumors, hematologic malignancies, and viral infections.

[0372] Tumors for which this treatment method is useful include any malignant cell type, such as those found in solid tumors or hematologic malignancies. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of: pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematologic malignancies include tumors of the bone marrow, T- or B-cell malignancies, leukemia, lymphoma, germ cell tumor, myeloma, etc. Further examples of cancers that can be treated using the methods presented 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, stomach or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), 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.

[0373] The cancer may specifically be, but is not limited to, the following histological types: growths, malignant; carcinoma; undifferentiated carcinoma; giant and fusiform cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilonidal stromal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous polypoid adenocarcinoma; adenocarcinoma, familial adenomatous polyposis; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic cell carcinoma; eosinophilic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granulocytic carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-capsulated Sclerosing carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Skin adnexal carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cystic adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous gland carcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma w / squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Theca cell tumor, malignant; Granulosarcoma, malignant; Androcytoma, malignant; Setori cell carcinoma; Ledich cell tumor, malignant; Lipiocytoma, malignant; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Hemangiosarcoma; Malignant melanoma Tumors; amelanoma; superficial diffuse melanoma; malignant lentigines melanoma; acral lentigines melanoma; nodular melanoma; malignant melanoma within a giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; small vesicular rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; Miller mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner's tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroscleroma, Malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; subcortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pineal tumor, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primary neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma;Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granulocytoma, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's disease; Granuloma-like tumor; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specialized non-Hodgkin's lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin's lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate / follicular NHL; Intermediate diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cleaved cell NHL; Giant cell NHL; Mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; malignant histiocytic hyperplasia; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel 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.

[0374] In some embodiments of this disclosure, immune cells are delivered to an individual in need, such as an individual with cancer or an infection. The cells then enhance the individual's immune system to attack the respective cancerous or pathogenic cells. In some cases, the individual is provided with one or more doses of the immune cells. In cases where the individual is provided with two or more doses of the immune cells, the duration between administrations should be sufficient to allow time for proliferation within the individual, and in particular embodiments, the duration between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[0375] Certain embodiments of this disclosure provide methods for treating or preventing immune-mediated conditions. 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 glands, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenic purpura, Behçet's disease, bullous pemphigoid, cardiomyopathy, fulminant dermatitis of the abdomen, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Thye-Stokes syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyalgia, glomerulonephritis, Graves' disease, Gibbs-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile delinquency. 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., minimal change disease, focal glomerulosclerosis, or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis 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-person syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (e.g., polyarteritis nodosa, high-stress arteritis, temporal arteritis / giant cell arteritis, or herpes-like dermatitis, vasculitis), leukoplakia, 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 1 diabetes mellitus, Crohn's disease, ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The subjects may also have allergic conditions such as asthma.

[0376] In another implementation, the subject is the recipient of the transplanted organ or stem cells, and immune cells are used to prevent or treat rejection. In a particular implementation, the subject has graft-versus-host disease (GVHD) or is at risk of developing GVHD. GVHD is a possible complication of any graft that uses or contains stem cells from a related or unrelated donor. There are two types of GVHD: acute and chronic. Acute GVHD occurs within the first three months after transplantation. Symptoms of acute GVHD include a small, reddish rash on the hands and feet, which may spread and become more severe, with skin peeling and blistering. Acute GVHD can also affect the stomach and intestines, in which case painful cramps, nausea, and diarrhea are present. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD has affected 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; however, chronic GVHD may also affect the mucous glands in the eyes, the salivary glands in the mouth, and the glands that lubricate the stomach lining and intestines. Any of the immune cell populations disclosed herein can be used. Examples of transplanted organs include solid organ grafts such as kidney, liver, skin, pancreas, lung, and / or heart, or cell grafts such as islets of Langerhans, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The grafts may be composite grafts, such as facial tissue. Immune cells can be administered before, concurrently with, or after transplantation. In some embodiments, the immune cells are administered before transplantation, for example, 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 a particular, non-limiting example, the administration of a therapeutically effective amount of immune cells occurs 3-5 days prior to transplantation.

[0377] In some implementations, the subject may be given non-myeloablative lymphocyte depletion chemotherapy prior to immunotherapy. The myeloablative lymphocyte depletion chemotherapy can be any suitable such therapy, which can be administered via any suitable route. The myeloablative lymphocyte depletion chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which may be metastatic. An exemplary route of administration of cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine can be administered. In a particular aspect, approximately 60 mg / kg of cyclophosphamide is administered for two days, followed by approximately 25 mg / kg of cyclophosphamide. 2 Fludarabine for five days.

[0378] In some embodiments, a growth or differentiation factor that promotes the growth, differentiation, and activation of the immune cells is administered to the subject, either concurrently with or after the immune cells. The immune cell growth factor can be any suitable growth factor that promotes the growth and activation of the 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, such as 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.

[0379] Therapeutic amounts of immune cells can be administered via a variety of routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, intracardiac, intrathecal, or intra-articular injection or infusion.

[0380] The therapeutically effective amount of immune cells used in adoptive cell therapy is the amount that achieves the desired effect in a subject undergoing treatment. For example, this could be an amount of immune cells necessary to suppress the progression of or induce the remission of autoimmune or alloimmune diseases, or to alleviate symptoms caused by autoimmune diseases, such as pain and inflammation. It could be an amount necessary to alleviate symptoms associated with inflammation, such as pain, edema, and fever. It could also be an amount necessary to reduce or prevent rejection of the transplanted organ.

[0381] The immune cell population can be administered according to a treatment regimen consistent with the disease, such as single or several doses over one to several days to improve the disease status, or regular doses over an extended period to suppress disease progression and prevent relapse. The precise dosage used in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be determined based on the physician's judgment and the individual patient's situation. The therapeutically effective dose of immune cells will depend on the subject undergoing treatment, the severity and type of the disease, and the method of administration. In some embodiments, the dose that can be used in the treatment of human subjects starts from at least 3.8 × 10⁻⁶. 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 2Variations are made. In some implementations, the dose used in the treatment of human subjects varies from approximately 3.8 × 10⁻⁶. 9 Up to approximately 3.8 × 10 10 immune cells / m 2 The dosage can be modified. In another implementation, the therapeutically effective dose of immune cells can be increased from approximately 5 × 10⁻⁶. 6 Cells / kg body weight up to approximately 7.5 × 10⁻⁶ 8 Cells / kg body weight, for example, approximately 2 × 10⁻⁶ 7 1 cell to approximately 5 × 10 8 Cells / kg body weight, or approximately 5 × 10⁻⁶ 7 1 cell to approximately 2 × 10 8 The number of immune cells varies per kg of body weight. The accurate amount of immune cells can be readily determined by those skilled in the art based on the subject's age, weight, sex, and physiological condition. The effective amount can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0382] The immune cells may be administered in combination with one or more other therapeutic agents for treating immune-mediated conditions. Combination therapies may include, but are not limited to: one or more antimicrobial 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), immune-depleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (e.g., azathioprine, or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids such as hydrocortisone, dexamethasone, or prednisone, or nonsteroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (e.g., interleukin-10 or transforming growth factor-β), hormones (e.g., estrogens), or vaccines. Additionally, immunosuppressive or tolerance-inducing agents may be administered, including but not limited to: calcium-dependent phosphatase inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil; antibodies (e.g., those recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, trooseltamivir, busulfan); radiation; or chemokines, interleukins, or inhibitors thereof (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 the immune cells, depending on the desired effect. The administration of the cells and the agents may be via the same or different routes, and at the same or different sites.

[0383] A. Pharmaceutical composition

[0384] Pharmaceutical compositions and formulations comprising an unlimited number of immune cells (e.g., T cells or NK cells) and pharmaceutically acceptable carriers are also provided herein.

[0385] The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (e.g., an antibody or peptide) with the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 22nd edition, 2012), in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids). Examples of pharmaceutically acceptable carriers include glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (…). Baxter International, Inc. Certain exemplary sHASEGPs and methods of use (including rHuPH20) are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0386] B. Combination Therapy

[0387] In some embodiments, the compositions and methods of this embodiment involve a population of immune cells in combination with at least one additional therapy. The additional therapy may be radiotherapy, surgery (e.g., tumor resection and mastectomy), chemotherapy, targeted therapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0388] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an anti-transfer agent. In some embodiments, the additional therapy is the administration of a side-effect limiting agent (e.g., an agent designed to reduce the occurrence and / or severity of treatment side effects, such as an anti-nausea agent). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma radiation. In some embodiments, the additional therapy 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 therapy may be one or more of the chemotherapeutic agents known in the art.

[0389] Immunotherapy can be administered before, during, after, or in various combinations with other cancer therapies (e.g., immune checkpoint therapy). The administration can be at intervals ranging from simultaneous to minutes to days or weeks. In embodiments where immunotherapy is administered to the patient separately from other therapeutic agents, it is generally ensured that there is no significant time gap between each delivery so that the two compounds will still be able to exert a beneficial combined effect on the patient. In such cases, it is considered that the antibody therapy and anticancer therapy can be administered to the patient within approximately 12 to 24 or 72 hours of each other, and more particularly within approximately 6 to 12 hours of each other. In some cases, it may be desirable to significantly extend the duration of treatment, with intervals from several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) between administrations.

[0390] Various combinations can be used. In the example below, immune cell therapy is "A," while anti-cancer treatment is "B":

[0391] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / BB / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A.

[0392] Administration of any compound or therapy according to this embodiment to a patient will follow a general protocol for the administration of such compounds, which takes into account the toxicity of the agent, if any. Therefore, in some embodiments, there are steps for monitoring toxicities attributable to the combination therapy.

[0393] 1. Chemotherapy

[0394] A wide variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. The term "chemotherapeutic agent" is used to mean a compound or composition administered in cancer treatment. These agents and drugs are classified by their mode of activity within cells (e.g., whether and at what stage they affect the cell cycle). Alternatively, agents can be characterized based on their ability to directly crosslink DNA, embed themselves in DNA, or induce chromosomal and mitotic aberrations by influencing nucleic acid synthesis.

[0395] Examples of chemotherapy agents include: alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, inprossurfan, and piperazine; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; azacyclopropanes and methylamelamines, including hexamethylmelamine, triethylmelamine, triethylphosphamide, triethylthiophosphamide, and trihydroxymethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); lichen inhibitors... Callystatin; CC-1065 (including its synthetic analogues, callystatin, callystatin, and pyrazin); cryptophytin (especially cryptophytin 1 and cryptophytin 8); dolastatin; pyrazinamide (including synthetic analogues, KW-2189 and CB1-TM1); arugulatin; pancratistatin; sarcodictyin; spongiform septicemia; nitrogen mustards, such as chlorambucil, naphthylambucil, cyclophosphamide, estradiol, ifosfamide, nitrogen mustard, oxynitrogen mustard hydrochloride, melphalan, neonitrogen mustard, benzethonol, prednimustine, trazophosphatamide, and uracil mustard; nitrosoureas, such as carmustine, chloramphenicol, formustine, lomustine, and nimosine. Sine and ramustine; antibiotics, such as enematic antibiotics (e.g., calcipomycin, especially calcipomycin γ1I and calcipomycin ωI1); enematic anthracycline antibiotics, including enematic anthracycline antibiotic A; bisphosphonates, such as clophosphonates; esporamycin; and new carcinogen chromophores and related chromogens, enematic antibiotic chromophores, aclarubicin, actinomycin, authramycin, diazoserine, bleomycin, actinomycin C, carabicin, erythromycin, carcinogen, chromomycin, actinomycin D, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, doxorubicin (including morpholine doxorubicin, cyanomorpholine doxorubicin, 2-pyrrolidone) Drugs containing: doxorubicin and deoxydoxorubicin, epirubicin, isorubicin, idarubicin, maceralomycin, mitomycin C, mycophenolate mofetil, nogamycin, olivomycin, pepromycin, purulentin, doxorubicin, rodorubicin, streptozotocin, streptozotocin, tuberculin, ubenmex, fenestrated statin, and zolrubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, pteroxate, and trimethyltraxa; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and fluorouridine.Androgens, such as calotestosterone, drotalbuterol propionate, cyclothionol, meandrazan, and testrolide; antiadrenergic drugs, such as mitotane and tramostan; folic acid supplements, such as folinic acid; acetoglucan lactone; aldehyde phosphoramide glycoside; 5-aminolevulinic acid; enturacil; acridine; bestrabucil; bifenthrin; edatrazine; desphosphonamide; colchicine; desaccharin; elformithine; eletyl acetate; epoxigin; etoradin; gallium nitrate; hydroxyurea; lentinan; chlorhexidine Damage; methanotrophs, such as maytansine and anthraquinone; mitoxantrone; mitoxantrone; moperadol; nitroxacin; pentostatin; phenamet; pirarubicin; loxoantrone; podophyllotoxin; 2-ethylhydrazine; procarbazine; PSK polysaccharide complex; razorazine; lizoxin; cizonan; germanospiramine; Alternaria ketoacid; triaminoquinone; 2,2′,2″-trichlorotriethylamine; trichothecene compounds (especially T-2 toxin, verracurin). (in) A, Bacitracin A and Serpentin); Urethane; Vinpocetine; Dacarbazine; Mannomustine; Dibromomannitol; Dibromoeutherol; Piperobromide; Gacytosine; Arabin (“Ara-C”); Cyclophosphamide; Taxanes, such as paclitaxel and docetaxel; Gemcitabine; 6-Thioguanine; Mercaptopurine; Platinum coordination complexes, such as cisplatin, oxaliplatin and carboplatin; Vincristine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Long Vincristine; vinorelbine; novordino; teniposide; edaraxacum; donomycin; aminopterin; capecitabine; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin; procarbazine; purcamycin; gemcitabine; novordino; farnesyl-protein transferase inhibitors; antiplatinum; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0396] 2. Radiation therapy

[0397] Other factors that cause DNA damage and have been widely used include the targeted delivery of gamma rays, X-rays, and / or radioactive isotopes to tumor cells, commonly referred to as gamma rays, X-rays, and / or radioactive isotopes. Other forms of DNA damage have also been considered, such as microwaves, proton beam radiation, and UV radiation. Most likely, all of these factors cause a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dosage ranges for X-rays vary widely, from daily doses of 50 to 200 roentgens over extended periods (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dosage ranges for radioactive isotopes vary extensively and depend on the isotope's half-life, the intensity and type of radiation emitted, and the uptake by the proliferating cells.

[0398] 3. Immunotherapy

[0399] Those skilled in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods and compositions of this disclosure. In the context of cancer treatment, immunotherapeutic agents typically rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab This is one such example. Immune effectors can be, for instance, antibodies specific to certain markers on the surface of tumor cells. The antibody alone can act as an effector of a therapy, or it can recruit other cells to actually influence cell killing. The antibody can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and act as a targeting agent. Alternatively, the effector can be a lymphocyte carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells, NKT cells, innate lymphoid cells, and NK cells.

[0400] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) covalently linked to a cytotoxic drug and can be used in combination therapies. This approach combines the high specificity of the MAb targeting its antigenic target with a highly potent cytotoxic drug, resulting in a “armed” MAb that delivers its payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, leading to reduced toxicity and an improved therapeutic index. Exemplary ADC drugs include… (brentuximabvedotin) and (trastuzumab emtansine or T-DM1).

[0401] In one aspect of immunotherapy, tumor cells must carry markers that are easily targeted, i.e., not present on most other cells. Many tumor markers exist, and any one of them 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 combining anticancer effects with immunostimulatory effects. Immunostimulatory molecules also exist, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, and γ-IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

[0402] Examples of immunotherapy include: immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapies, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF; gene therapies, such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185. It is considered that one or more anticancer therapies may be used in conjunction with the antibody therapies described herein.

[0403] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either upregulate (e.g., co-stimulatory molecules) or downregulate. 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 T-cell activation V-domain Ig inhibitor (VISTA). In particular, the immune checkpoint inhibitor targets the PD-1 axis and / or CTLA-4.

[0404] The immune checkpoint inhibitors may be drugs, such as small molecules, recombinant forms of ligands or receptors, or particularly antibodies, such as human antibodies. Known inhibitors of immune checkpoint proteins or their analogues may be used, particularly chimeric, humanized, or human forms of antibodies. As those skilled in the art will appreciate, alternative and / or equivalent names may be used for certain antibodies mentioned in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0405] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In one particular aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In one particular aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In one particular aspect, the PDL2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

[0406] 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 an 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...) Pembrolizumab is a usable anti-PD-1 antibody. (Also known as MK-3475, Merck 3475, Lanlolizumab, etc.) SCH-900475 is an example of an 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 PD-L2-Fc fusion soluble receptor.

[0407] Another immune checkpoint that can be targeted using the methods presented in this paper 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 it binds 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 is similar to the T-cell costimulatory protein CD28, and 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, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and is likely important for their function. T cell activation via T cell receptors and CD28 leads to increased expression of CTLA-4 (an inhibitory receptor for the B7 molecule).

[0408] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

[0409] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in this method can be generated using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and...). Or its antigen-binding fragments and variants. In other embodiments, the antibody comprises the heavy and light chain CDRs or 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 with the antibodies mentioned above for binding to CTLA-4 and / or binds to the same epitopes on CTLA-4 as the antibodies mentioned above. In yet another embodiment, the antibody has at least approximately 90% variable region amino acid sequence identity with the antibodies mentioned above (e.g., at least approximately 90%, 95%, or 99% variable region identity with ipilimumab).

[0410] 4. Surgical procedures

[0411] Approximately 60% of people with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgeries. Curative surgeries include resections in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can be used in combination with other therapies, such as the treatments described in this protocol, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical procedures (Moss surgery).

[0412] After the removal of some or all cancerous cells, tissue, or tumors, a cavity may form in the body. Treatment can be performed by perfusion, direct injection, or local application of additional anticancer therapies to the area. Such treatments 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 may also have varying dosages.

[0413] 5. Other reagents

[0414] Other agents that can be used in combination with certain aspects of this embodiment to improve therapeutic efficacy have been considered. These additional agents include: agents that upregulate cell surface receptors and GAP connections, cell inhibition and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of highly proliferating cells to apoptosis-inducing agents, or other biological agents. Increased intercellular signaling resulting from increased GAP connections will increase the anti-hyperproliferative effect on adjacent highly proliferating cell populations. In other embodiments, cell inhibition or differentiation agents can be used in combination with certain aspects of this embodiment to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors have been considered for use to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Further consideration has been given to using other agents that increase the sensitivity of highly proliferating cells to apoptosis, such as antibody C225, in combination with certain aspects of this embodiment to improve therapeutic efficacy.

[0415] VII. Manufactured products or reagent kits

[0416] This document also provides manufactured articles or kits containing an unlimited number of immune cells. The manufactured articles or kits may further include a packaging insert containing instructions on using the immune cells to treat cancer in an individual or delay cancer progression or enhance the immune function of an individual with cancer. Any antigen-specific immune cells described herein may be included in the manufactured articles or kits. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a wide variety of materials such as glass, plastics (e.g., polyvinyl chloride or polyolefins) or metal alloys (e.g., stainless steel or Hastelloy corrosion-resistant nickel-based alloys). In some embodiments, the container contains the formulation and a label (which may indicate instructions for use) on or attached to the container. The manufactured articles or kits may further include other materials desired from a commercial or user standpoint, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. In some embodiments, the manufactured articles further include one or more additional reagents (e.g., chemotherapy reagents and antitumor reagents). Suitable containers for said one or more reagents include, for example, bottles, vials, bags, and syringes.

[0417] IV. Examples

[0418] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art will recognize that the techniques disclosed in the following embodiments represent techniques that the inventors have found to function well in the practice of the invention, and therefore can be considered as constituting preferred practices with respect to the invention. However, based on this disclosure, those skilled in the art will recognize that many variations can be made in the particular embodiments disclosed, and that they still yield the same or similar results without departing from the spirit and scope of the invention.

[0419] Example 1 – Unlimited Immune Cells for Adoptive Therapy

[0420] 293T cells were cultured and passaged in 10 mL high-glucose DMEM medium containing 10% FBS and 1% Pen / Strep in T75 flasks. Once the 293T cells reached 90% confluence, they were used for transfection the following day for lentiviral vector generation and plasmid packaging. The coding sequences of the BCL6 and Bcl-xL genes were ligated with the T2A sequence to generate an open reading frame that simultaneously expresses the BCL6 and Bcl-xL genes. This BCL6-T2A-Bcl-xL open reading frame was cloned into a lentiviral vector using Gibson assembly following the experimental protocol provided by NEB. The final vector was named pLV4a plasmid. Figure 1AThe pLV4a plasmid was co-transfected into 293T cells along with a lentiviral vector packaging mixture from abm. The viral supernatant was concentrated using a Lenti-X concentrator from Clontech.

[0421] To develop an unlimited cell line from a healthy donor, RosetteSep was used. TM Human T cell enrichment mixture and SepMate TM -50 tubes (from STEMCELL Technologies) were used to isolate normal T cells from healthy donors. The isolated T cells were then treated with a solution supplemented with 10% FBS, 2% HEPES, 1% sodium pyruvate, and 0.01% 2-mercaptoethanol, along with 50-1000 IU / mL IL-2 (Genscript) and 25 μL / mL ImmunoCult. TM Human CD3 / CD28 / CD2T cell activators (STEMCELL Technologies) were cultured in RPMI-1640 medium (Gibco). After 36–48 hours of culture, the cells were transfected with concentrated pLV4a lentiviral vector in the presence of RetroNectin (Clontech). Figure 1A One million cultured T cells were transduced, then cultured in RPMI 1640 medium in the presence of 50-1000 IU / mL IL-2, passaged, and separated (when needed). Some of the transduced T cells continued to proliferate indefinitely. This method generates a T cell line known as "unlimited T cells" from healthy donor T cells, which proliferates in the presence of recombinant human IL-2 or IL-15.

[0422] Next, several new unlimited T cell lines were generated using the methods described above. These were named In1-L4a T cells, which consist of multiple subtypes of T cells. Using In1-L4a T cells, a series of T cell lines were isolated and generated through cell sorting or genetic engineering, including Ie1-L4a, If1-L4a, In1-L4aJ3, Ie1-L4aJ3, Igd1-L4a, and Igd1-L4aJ3. Detailed descriptions of these IL-2 or IL-15-dependent unlimited T cell lines are summarized in Table 1.

[0423] Table 1. Unlimited available T cells

[0424]

[0425] In1-L4a and derived cells were cultured in a standard medium, such as one containing GlutaMax. TMMaintenance can be easily achieved in RPMI 1640 medium supplemented with sodium pyruvate and 10% fetal bovine serum (FBS). Additionally, for long-term growth, supplementation with 50-1000 IU / mL of recombinant human IL-2 (IL-2) is recommended. Figure 1B IL-15 also supports proliferation, but IL-7 or IL-21 do not. Figure 1B When the suspension culture was maintained by changing the culture medium every half week, the cells were able to proliferate and expand very rapidly in an exponential pattern, with a doubling time of approximately 24 hours. These unlimited T cells were kept in culture and continued to proliferate for over 3 months, with no change in the proliferation rate in the presence of IL-2. Figure 1B ).

[0426] The cells were highly dependent on IL-2 for survival and proliferation, and stopped proliferating and died rapidly after IL-2 was removed from the culture medium. Figure 1B The unlimited T cells are CD3 positive, while other surface markers such as CD4 or CD8, TCRαβ or TCRgδ or CD16 are expressed on some subclasses of unlimited T cells, even after long-term in vitro culture and expansion. Figure 1C Those markers indicate that the infinite number of T cells is a mixed population of different subtypes of T cells. Figure 1C Therefore, specific T cell populations can be isolated through cell sorting using specific T cell markers. For example, CD8+ unlimited T cells can be isolated through cell sorting using anti-CD8 antibodies. Another specific T cell population, the γδ T cell population, can also be isolated through cell sorting using anti-TCRgδ antibodies. After sorting, a relatively pure γδ T cell line is generated. Figure 1D ).

[0427] Mature T cells can further differentiate into different functional subclasses such as Th1, Th2, Th17, Treg, and Tfh in lymphoid tissues. Differentiation into these subclasses is driven by unique master transcription factors. For example, Th1 differentiation is driven by Tbet, Th2 by GATA-3, Th17 by RORgt, Treg by Foxp3, and Tfh 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 differentiation type has not been observed in unlimited T cells, which is unexpected.

[0428] The cells are further modified to express an anti-CD19 CAR, thereby generating a series of "anti-CD19 inCART cells". The CD3 inCART and CD8 inCART cells, In1-L4a and Ie1-L4a, are modified to express a chimeric antigen receptor (CAR) targeting human CD19 on their surface, using a vector named pJ3 plasmid. Figure 2A This leads to an unlimited number of In1-L4aJ3 and Ie1-L4aJ3 T cell lines. Both In1-L4aJ3 and Ie1-L4aJ3 T cells express anti-CD19 CAR and can bind to recombinant human CD19 protein. Figure 2B and 2C In1-L4aJ3 and Ie1-L4aJ3 unlimited T cells were successfully generated and expanded in vitro, exhibiting a proliferation rate similar to their parental cells. At effector:target ratios of 0.2:1 and 1:1, Ie1-L4aJ3 cells demonstrated the ability to lyse CD19-positive Raji lymphoma cell lines and Nalm6 leukemia cell lines in the presence of IL-2. Figure 3 ).

[0429] Example 2 – Modifying In1-L4a-derived T cell lines to generate CD19 in CART cells

[0430] The following examples describe the modification of In1-L4a-derived unlimited T cell lines to generate CD19 in CAR T cells. These procedures can be used similarly on other unlimited T cell lines; however, for simplicity, the procedures are described in detail only with respect to In1-L4a and Ie1-L4a cell lines. Those skilled in the art can adapt the methods to insert anti-CD19 CAR genes into other unlimited cell lines, or to insert other CARs or TCRs targeting different tumor markers, for therapeutic purposes against a wide variety of different tumors.

[0431] A recombinant lentiviral vector expressing anti-CD19 CAR and hEGFRt driven by the MSCV promoter was generated using the Gibson assembly method (NEB). This vector was named pJ3(LV-MSCV-optimized C19-CD28z-T2A-tEGFR). Figure 2AThe pJ3 plasmid and lentiviral vector packaging mixture (ABM) was co-transfected into 293T cells to generate infectious pJ3 virus. 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 an anti-EGFR antibody (R&D) labeled with AF647 and recombinant human CD19 protein (ACROBiosystems) labeled with FITC. The percentage of CAR-positive cells in the pJ3-transduced Ie1-L4a and In1-L4a groups was approximately 20% and 46.5%, respectively. Figure 2B ).

[0432] The percentage of CAR positivity was further confirmed by double staining with recombinant human CD19 protein labeled with FITC and cetuximab labeled with AF647. Figure 2C 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 targeting human CD19 were named In1-L4aJ3 and Ie1-L4aJ3, respectively. They exhibited an exponential proliferation rate similar to their parental In1-L4a and Ie1-L4a unlimited T cells. Figure 1B ).

[0433] 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 Burkitt lymphoma patients, widely used in preclinical lymphoma studies, while Nalm6 is a CD19+ B-cell leukemia cell line derived from acute lymphoblastic leukemia patients. Therefore, both can be used to test the cytotoxic activity of unlimited anti-CD19 CAR cell lines by co-culturing effector cells and target cells in the presence of IL-2 at ratios of 0.2:1 and 1:1. The assay was performed in 12-well plates. Briefly, 100,000 Raji or Nalm6 cells per well were cultured with 20,000 or 100,000 Ie1-L4aJ3 (anti-CD19 CAR) or Ie1-L4a (anti-CD19 CAR-free) cells in 2 mL of the aforementioned medium. After 5 days of co-culture, cells in each well were stained with an APC-conjugated anti-CD8 antibody (BD), and cells were harvested using a BD Fotessa analyzer (BD) to determine the percentage of viable T cells and tumor cells. Flow cytometry data were analyzed using FlowJo software. The data demonstrated that both types of Ie1-L4aJ3 unlimited T cells could effectively lyse Raji and Nalm6 tumor cells in vitro. Figure 3 Conversely, no significant lysis of Raji or Nalm6 tumor cells was observed with Ie1-L4a cells because they lacked anti-CD19 CAR.

[0434] Example 3 – Unlimited T Cells for Off-the-shelf Adoptive T Cell Therapy

[0435] Unlimited T cells have the ability to proliferate rapidly and over a long period of time. To date, we have generated unlimited T cells from eight healthy donors via lentiviral transduction of BCL6 and BCL2L1, and have observed that they can grow rapidly and continuously for >12 months in the presence of IL-2 or IL-15. Incorporation of anti-CD19 CAR into these cells via lentiviral transduction did not affect their growth rate. The fold increase in these T cells was ~100-fold at 10 days and ~1 million-fold at 30 days, and their proliferative capacity remained unchanged during 12 months of continuous in vitro culture (Figure 5A). Phenotypic, these unlimited T cells are derived from CD4+. + and CD8 + The mixture of T cells can be sorted to high purity using magnetic beads (Figure 5B). In CD4 + Foxp3 in T cells +Cells <5% (data not shown). Removal of cytokines at any point resulted in rapid cell death within one week, suggesting that these T cells had not yet transformed into a malignant phenotype and had not developed the ability to grow autonomously (Figure 5C).

[0436] Unlimited T cells exhibit high telomerase activity Since T cell proliferation after 30-40 population doublings leads to gradual telomere shortening and replicative senescence (Barsov et al., 2011), the inventors measured telomerase activity in these cells using the TRAPEze Telomerase Activity Assay Kit (Sigma). hTERT activity in the infinite T cells was very high relative to the corresponding T cells from peripheral blood mononuclear cells (PBMCs) (Fig. 6A). RNA-seq analysis of these cells was consistent with this observation in infinite CD4+, infinite CD8+, and infinite CD8+CAR+ T cells (Fig. 6B). These results suggest that transduced genes may lead to high telomerase activity in infinite T cells, resulting in telomere length stabilization, prevention of replicative senescence, and the conferring of long-term proliferative capacity.

[0437] Incorporation of anti-CD19 CAR alters the specificity of unlimited T cells against B-cell malignancies. Transducing anti-CD19CAR (based on clone FMC63, anti-CD19scFv, which possesses a CD8α hinge / transmembrane domain, CD3ζ and CD28 signaling domains, and tEGFR as a transduction marker and safety switch (Wang et al., 2011)) into unlimited T cells via lentivirus enabled them to efficiently and specifically degranulate and kill Daudi Burkitt lymphoma and NALM-6 acute B-cell lymphoblastic leukemia cell lines (Figs. 7A-7B). Unlimited T cells without CAR did not show any significant cytotoxicity or degranulation. Unlimited T cells were slower in killing tumor cells than conventional CAR T cells generated from freshly isolated T cells from healthy donors, but they were almost completely eliminated by day 7 (Fig. 7A). This slower killing may be a potential advantage in clinical practice as it may induce fewer toxicities, such as cytokine release syndrome and neurotoxicity. These anti-CD19 unlimited CAR T cells exhibit central and effector memory phenotypes (Fig. 7C) and express very low or no markers associated with T-cell exhaustion (Fig. 7D).

[0438] Transcriptional profile of unlimited T cells Compared to the corresponding CD4 isolated from PBMC samples. + or CD8 + For T cells, the presence or absence of anti-CD19 CARs and unlimited CD4+ + and / or CD8 +RNA-seq analysis of T cells was consistent with flow cytometry and functional data, indicating that these cells possessed memory and cytotoxic phenotypes and did not express markers associated with classic T cell exhaustion (Figures 8A-8B). Although they differentiated from naive T cells into follicular helper T cells (T cells) through overexpression of BCL6 (a marker related to T cell differentiation), this did not lead to T cell exhaustion. FH ) main transcription factor 3 These cells are generated from T cells, but they do not exhibit T cells. FH The signature (Fig. 8A) does not express a high level of CXCR5 (Fig. 8C), which is T FH Cellular markers (Nurieva et al., 2009; Rawal et al., 2013). However, they retain expression of chemokine receptors (CCR4 and CCR7, important for transporting T cells to lymph nodes, and CXCR4, important for transport to bone marrow) (Fig. 8C) (Viola et al., 2006); both sites are frequently involved in lymphoma. The unlimited T cells do not express aging markers such as B3GAT1 (CD57), CD160, or KLRG1 (Fig. 8D) (Xu et al., 2017). The gene expression profiles of chemokines (Fig. 9A) and cytokines (Fig. 9B) are largely similar between unlimited T cells and their corresponding CD4 or CD8 T cells derived from peripheral blood. Cytokine receptor gene expression showed some differences, including but not limited to increased levels of IL2RA, IL15RA, and IL21R in unlimited T cells compared to corresponding CD4 or CD8 T cells derived from peripheral blood, and decreased levels of IL4R, IL7R, IL10RA, IL17RA, IL18R1, and IFNGR1 (Figure 9C).

[0439] Unlimited CAR T cells retain proliferative and cytotoxic functions after cryo-thawing. Unlimited T cells with and without CAR were cryopreserved and thawed after 6 months. Upon thawing, they showed strong CAR expression, with the use of an anti-EGFR antibody (Fig. 10A). Culture of these cells in IL-2 showed a ~100-fold increase in cell number over 10 days, confirming that the proliferative capacity of the said unlimited CD8 CAR T cells was maintained after freeze-thaw (Fig. 10B). Furthermore, these cells exhibited highly significant and specific cytotoxic activity against malignant B cells (Fig. 10C).

[0440] Unlimited γδ T cells do not express exhaustion markers Unlimited γδ T cells do not significantly express classic markers of T cell exhaustion. Figure 11 ).

[0441] Anti-CD19 unlimited CAR T cells demonstrated anti-tumor efficacy in in vivo models.By using unlimited CAR T cells labeled with luciferase, the inventors observed that, upon 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 (middle column), which may be because mouse cytokines (both IL-2 and IL-15) do not support the growth of human T cells. Conversely, injection of recombinant human IL-15 on days 1 and 3 induced substantial T cell proliferation, with cells persisting for up to one week after IL-15 cessation ( Figure 12 (Right column). These results suggest that IL-15 promotes proliferation and persistence in vivo, but low doses may be sufficient. Similar effects were observed with IL-2.

[0442] Next, the inventors added NALM-6 tumor cells labeled with luciferase along with 3 × 10 6 Unlimited T cells / mouse (with or without CAR) were administered intravenously (IV) to NSG mice, with IL-15 injected on days 0, 4, 7, and 11. Significant tumor control and prolonged survival were observed in mice treated with unlimited CAR T cells compared to those without CAR. Figure 13 In summary, these results provide a theoretical basis for modifying unlimited T cells to secrete IL-2 or IL-15, thereby enhancing their in vivo expansion and persistence.

[0443] Microbe-associated and tumor-associated antigen-specific unlimited T cells Tests using tetramers on an unlimited number of T cells generated from HLA-A2+ donors revealed the presence of a mixture of microbe- and tumor-associated antigen-specific T cells. Figure 14 To generate rich clusters of these T cells, the inventors stimulated peripheral blood mononuclear cells from healthy donors with a peptide aggregate derived from EBV proteins. After 24 hours, CD137-positive T cells were sorted and used to generate an unlimited number of T cells, which were transduced using the lentiviral vector L5x expressing BCL6 and BCL2L1. Figure 22The viral production and transduction protocol is described in Example 1. Two weeks after transduction, the transduced T cells were restimulated with CD3 / CD28 / CD2 T cell activators and then 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 by APC enrichment beads. The enriched unlimited T cells were then cultured as all other unlimited T cells. At week 13, the enriched unlimited T cells were stained with the APC-labeled BMLF1-HLA-A2 tetramer, and approximately 70% of the T cells were found to be CD8 positive and BMLF1-HLA-A2 tetramer positive, suggesting that they are specific to the HLA-A2 binding peptide (GLCTLVAML) derived from the EBV-BMLF1 protein. Figure 15 Similar methods can be used to generate other antigen-specific T cells targeting microorganisms and tumor-associated antigens. Such antigen-specific T cells can then be used to transduce targeted CARs or TCRs to generate dual antigen-specific T cells.

[0444] Tet-off system as a safety switch The inventors did not observe any malignant transformation of unlimited T cells or cytokine-independent growth in vitro, even in cultures of unlimited T cells derived from 8 donors for 6 to >12 months. Figure 4 However, to ensure safety regarding clinical conversion, a Tet-off safety switch was incorporated, which allows us to shut down transduced BCL6 and BCL2L1 genes using doxycycline. After incorporation of this Tet-off safety switch, unlimited T cells maintained their growth rate in the absence of doxycycline, but ceased proliferation and underwent gradual cell death in the presence of 1 μg / mL doxycycline (the concentration achievable with the standard therapeutic dose of doxycycline in humans (Agwuh et al., 2006)). Figure 16 Optical microscopy imaging revealed that unlimited T cells gradually decreased in size with increasing doxycycline concentrations, along with a reduction in the number of cells with proliferative clusters. Figure 17 In addition, CD25 expression was significantly reduced in the presence of doxycycline. Figure 17 Increased PD-1 expression suggests that BCL6 and / or BCL2L1 genes may control the expression of these molecules. The expression of other T-cell co-inhibitory receptors was not significantly altered in the presence of doxycycline. Figure 18 A similar tet-off safety switch could also be used to control the incorporation of IL-2 or IL-15 cytokine genes into unlimited T cells.

[0445] Anti-CD19 unlimited CAR T cells respond to B-cell tumor cells by producing effector cytokines To determine the cytokine profile of unlimited T cells produced in response to tumor cells, the inventors mixed NALM-6 tumor cells with CD8 cells transduced with or without anti-CD19 CAR at a 5:1 effector:target ratio. + Unlimited T cells were co-cultured together. Cytokine levels were measured in the supernatant after 3 days. Results showed that unlimited T cells with anti-CD19 CAR (but not without anti-CD19 CAR) responded to NALM-6 tumor cells by primarily producing significant amounts of IL-2, GM-CSF, IFNγ, IL-5, and IL-17. Figure 19 The production of TNFα, IL-4, IL-6, IL-10, or IL-13 in response to tumor cells by anti-CD19 unlimited CAR T cells is minimal or not significantly different from that of unlimited T cells without CAR expression. However, unlimited T cells with or without CAR expression produce large amounts of IL-4, exceeding 10,000 pg / mL, in the presence or absence of tumor cells. Figure 19 (And data not shown). The property of unlimited T cells constitutively producing large amounts of IL-4 in the absence of external stimuli may have potential clinical applications for treating a variety of inflammatory conditions, such as autoimmune diseases, graft-versus-host disease, certain types of infections associated with cytokine release syndromes, toxicity associated with CAR T-cell and other adoptive T-cell therapies, inflammatory bowel disease, immune-related adverse events associated with various immunotherapies, hemophagocytic lymphohistiocytosis, periodic fever syndrome, etc., because IL-4 can suppress inflammation induced by T cells, macrophages, and other immune cells.

[0446] tEFGR safety switch for anti-CD19 unlimited CAR T cells To determine whether truncated EGFR (tEGFR) could act as a safety switch for unlimited T cells, the inventors co-cultured unlimited T cells expressing anti-CD19 CAR and tEGFR with or without natural killer (NK) cells isolated from peripheral blood mononuclear cells from healthy donors in the presence of cetuximab at a concentration of 5 μg / mL. Compared to rituximab used as a control, cetuximab induced significant lysis of anti-CD19 unlimited CAR T cells via antibody-dependent cell-mediated cytotoxicity (ADCC). Figure 20 These results suggest that tEGFR could act as a safety switch to eliminate an unlimited number of T cells in the body in the event of adverse events.

[0447] Generate unlimited T cells by transducing BCL6 and BIRC5 genes.The inventors observed that unlimited T cells can be generated by transducing the BCL6 and BCL2L1 genes or by transducing the BCL6 and BIRC5 genes into human T cells (Figure 21A). BCL2L1 encodes Bcl-xL, an anti-apoptotic protein; while BIRC5 encodes a survival protein, a family of inhibitors of apoptosis (IAP) proteins that promote proliferation and block apoptosis in cells. Transduction of either gene combination results in the generation of unlimited T cells with considerable long-term proliferative potential at an exponential growth rate in the presence of IL-2 (Figure 21B). Furthermore, these unlimited T cells are generated using a teet-off safety switch that allows us to turn off the transduced BCL6 and BCL2L1 or BCL6 and BIRC5 genes by using doxycycline. The vector also incorporates the IL-15 gene, which is transduced into these cells. The cells grew exponentially in the absence of doxycycline, but ceased proliferation and underwent gradual cell death in the presence of 1 μg / mL doxycycline, despite transduction of IL-15 and the addition of IL-2 to the culture medium (Fig. 21C).

[0448] An example of a construct containing BCL6 and Bcl-xl is L5x(MSCV-BCL6-P2A-BCL-xl-T2A-rtTA). This structure includes at least wild-type BCL-6 separated from BCL-xL by a P2A element, and BCL-xL separated from rtTA (Tet on trans-activator) by a T2A element.

[0449] Figure 23 Several examples of implementation schemes including at least BCL6 are provided; such examples may or may not use BCL-xL. By way of example only, Example 1 uses the MSCV promoter to regulate BCL6 and rtTA overexpression, and the H1 promoter regulates a shRNA targeting caspase 9 to knock down caspase 9 expression. Example 2 uses the MSCV promoter to regulate BCL6 and rtTA overexpression, except that it uses the human U6 promoter to regulate a shRNA targeting the BAK gene to knock down BAK expression. In Example 3, the MSCV promoter regulates BCL6 and HSP27 and rtTA overexpression. In Example 4, the MSCV promoter regulates BCL6 and rtTA expression, and the U6 promoter regulates miRNA21 expression.

[0450] All methods disclosed and claimed herein can be made and performed based on this disclosure without excessive experimentation. While the compositions and methods of the invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be made to the methods described herein and to the steps or sequence of steps thereof without departing from the concept, spirit, and scope of the invention. More particularly, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein with the same or similar results. All such similar substitutions or modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention, as defined by the appended claims.

[0451] References

[0452] The following references are specifically incorporated herein by reference to provide exemplary procedures or other details that are supplementary to those set forth herein.

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[0459] Nurieva RI, Chung Y, Martinez GJ et al., Bcl6 mediates the development of T follicular helper cells. Science. 2009, 325(5943): 1001 - 1005.

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Claims

1. A composition comprising T cells modified to overexpress (a) BCL6 and Bcl-xL, or (b) BCL6 and a surviving protein, wherein the T cells are modified to express one or more chimeric antigen receptors (CARs).

2. The composition of claim 1, wherein the T cells produce IL-4 in the absence of external stimulation.

3. The composition of claim 1, wherein the T cells are modified to express IL-2 and / or IL-15.

4. The composition of claim 1, wherein the T cells are derived from a donor who has not been diagnosed with cancer.

5. The composition of claim 1, wherein the T cells are derived from an individual in need of treatment.

6. The composition of claim 4, wherein the donor is a person.

7. The composition of claim 5, wherein the individual is a person.

8. The composition of claim 1, wherein the T cell is a CD4+ T cell, CD8+ T cell, NKT cell, γδ T cell or a combination thereof.

9. The composition of claim 1, wherein the T cell is a naive T cell, an effector T cell, a memory T cell, or a combination thereof.

10. The composition of claim 1, wherein the T cell is a TCR αβ cell.

11. The composition of claim 1, wherein the T cell is a T cell that is Th1 / Tc1, Th2 / Tc2, Th9 / Tc9, Th17 / Tc17, Tfh, Th22, Tc22 or a combination thereof.

12. The composition of claim 1, wherein the T cells express cytokines and cytotoxic molecules, which are IFNγ, GM-CSF, TNFα, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-16, IL-17, IL-32, granzyme B, perforin, or combinations thereof.

13. The composition of claim 1, wherein the T cells are specific for one or more microbial antigens, one or more autoantigens, or one or more tumor antigens.

14. The composition of claim 1, wherein the T cells are modified to express one or more T cell receptors (TCRs).

15. The composition of claim 14, wherein the CAR and / or TCR targets the antigen-binding regions of CD19, CD20, CD22, CD79a, CD79b, mesothelin, MAGE-A1, MAGE-A4, TCL1, NY-ESO, WT1, and / or BAFF-R.

16. The composition of claim 14, wherein the CAR comprises a complete sequence from the following: a hinge of CD8a, CD28, PD-1, CTLA4, an α, β, or ζ chain of a T-cell receptor, CD2, CD3ζ, CD3ε, CD3γ, CD3δ, 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, TIM-3, HVEM, LIGHT, DR3, CD30, CD224, CD244, SLAM, CD226, DAP, or combinations thereof or synthetic molecules thereof.

17. The composition of claim 14, wherein the CAR comprises a complete transmembrane domain derived from: the α chain of a T-cell receptor, the β chain of a T-cell receptor, the ζ chain of a T-cell receptor, CD28, CD2, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, PD-1, CTLA4, DAP, synthetic molecules thereof, or combinations thereof.

18. The composition of claim 14, wherein the CAR comprises one or more co-stimulatory domains derived from CD28, CD27, OX-40 (CD134), DAP10, DAP12, 4-1BB, or a combination thereof.

19. The composition of claim 1, wherein the composition comprises 100,000 to 10 billion T cells.

20. The composition of claim 1, wherein the T cell comprises one or more safety switches.

21. The composition of claim 20, wherein the safety switch is a truncated EGFR or a fusion protein thereof.

22. The composition of claim 1, wherein the T cells express one or more growth factors, one or more differentiation factors, or a combination thereof.

23. The composition of claim 1, wherein the T cells maintain a proliferation rate of at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months or longer.

24. The composition of claim 1, wherein the T cells have enhanced antitumor cytotoxicity, cytokine production, in vivo proliferation, and / or in vivo persistence.

25. A method for generating T cells in vitro as defined in any one of claims 1-18 and 20-24, the method comprising introducing one or more vectors encoding (a) BCL6 and Bcl-xL, or (b) BCL6 and a survival protein into the T cells.

26. The method of claim 25, wherein the vector links BCL6 and Bcl-xL to the 2A sequence.

27. The method of claim 25, wherein the vector is a lentiviral vector.

28. The method of claim 25, wherein the introduction includes transducing the T cells with a lentiviral vector in the presence of IL-2 and / or IL-15.

29. The method of claim 28, wherein IL-2 is at a concentration of 10 IU / mL to 1000 IU / mL.

30. The method of claim 29, wherein IL-2 is at a concentration of 400 IU / mL.

31. The method of claim 25, further comprising activating the T cells with CD3 and CD28.

32. The method of claim 25, further comprising culturing the T cells in the presence of IL-2 and / or IL-15.

33. The method of claim 32, wherein IL-2 or IL-15 is present at a concentration of 10-200 ng / mL.

34. The method of claim 25, wherein the T cells are cultured for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months.

35. The method of claim 25, further comprising sorting T cell subclasses.

36. The method of claim 35, wherein the T cell subclass includes CD4+ T cells, CD8+ T cells, or γδ T cells.

37. The method of claim 25, further comprising introducing one or more cytokines and / or one or more safety switches into the T cells.

38. The method of claim 37, wherein the one or more cytokines and / or one or more safety switches are on the same vector as the (a) BCL6 and Bcl-xL, or the (b) BCL6 and survival protein genes.

39. The method of claim 37, wherein the one or more cytokines and / or one or more safety switches are on a vector different from that of (a) BCL6 and Bcl-xL, or (b) BCL6 and the survival protein gene.

40. Use of T cells as defined in any one of claims 1-18 and 20-24 in the preparation of a medicament for treating a disease in a subject.

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