LAT-activated chimeric antigen receptor T cells and methods of use thereof

Abstract

The present disclosure describes T cells expressing a chimeric antigen receptor (CAR), as well as pharmaceutical compositions comprising the T cells, and methods of making and using such T cells. In particular, the present disclosure describes T cells expressing a first CAR that binds to a first antigen and a second CAR that comprises a LAT intracellular signaling domain that binds to a second antigen, and methods of use in the treatment of cancers, such as solid tumors and hematological malignancies.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 321,549, filed March 18, 2022, and U.S. Provisional Patent Application No. 63 / 229,344, filed August 4, 2021, each of which is incorporated by reference in its entirety herein.

[0002] government support This invention was made with Government support under Grant No. K12CA086913-20 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] The present invention relates generally to the fields of molecular biology, immunology, oncology and medicine. More specifically, the present invention relates to immune cells expressing chimeric antigen receptors, such as chimeric antigen receptors, that bind to target proteins. [Background technology]

[0004] Over the past decade, chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy against B-lineage leukemia, lymphoma, and multiple myeloma, and is promising for the treatment of all malignancies that were incurable with conventional therapies. Through multiple clinical trials, CAR T cells targeting the CD19 antigen have induced complete remission in 70-90% of patients with multiple relapsed and / or refractory acute lymphoblastic leukemia (ALL). However, this remarkable upfront success does not translate into long-term remission for many patients, as longitudinal studies have shown that less than 50% of patients treated with CAR T cells remain in remission beyond one year after therapy due to post-CAR relapse. Post-CAR relapse remains a clinical challenge, as conventional chemotherapy, antibody-based therapies (blinatumomab and inotuzumab), and retreatment with the same CAR T cells have been found to rarely induce patients back into remission, the majority of which were short-lived.

[0005] CD19-directed CAR T-cell therapy for relapsed and / or refractory B-lineage lymphoma has shown similar results, with objective response rates (ORR) ranging from 52 to 82%, with 40 to 54% of patients achieving a complete response (CR), but disease relapse and / or progression after CAR T-cell therapy remains common, with fewer than 40% of patients remaining progression-free at 1 year. Consistent with the experience in leukemia, there are no established therapies that are effective for lymphoma patients whose disease has relapsed and / or progressed after administration of CAR T cells, and reinfusion of the same CAR T cells is largely ineffective.

[0006] Relapse after CAR therapy occurs through a variety of mechanisms. In B-cell leukemias treated with CD19-directed CAR T cells, initial treatment failure and relapse, in which the leukemia continues to express the CD19 antigen, are highly correlated with low levels of CAR T cell proliferation and short-term CAR T cell persistence in patients, and it is generally believed that improving CAR T cell proliferation and persistence will improve outcomes by preventing antigen-positive leukemia from relapsing. Another major mechanism of relapse after CAR T cell therapy is the modulation of the targeted antigen on malignant cells as a means of avoiding CAR T cell detection. In B-cell leukemia, this is mainly observed as the appearance of CD19-negative leukemia cells upon relapse. Similarly, reduced surface expression of the CD19 antigen in B-lineage lymphomas is associated with refractoriness to and relapse after treatment with CD19-directed CAR T cells. Either loss or downregulation of the antigen renders current CD19-directed CAR T cell therapies ineffective, making this outcome generalizable beyond CD19 to other CAR-targeted antigens.

[0007] To overcome relapse due to antigen modulation in leukemia / lymphoma, CARs targeting alternative antigens have been developed. CD22-directed CAR T cells have shown the ability to induce remission in 70-80% of patients with ALL, including those with CD19-negative relapse, after immunotherapy. Unfortunately, relapse was frequently observed in patients after CD22-directed CAR T cell therapy, mainly due to downregulation of the CD22 antigen. Currently, CD22 CAR T cell therapy is used to guide patients to consolidation hematopoietic stem cell transplantation (HSCT), but the long-term outcomes of this strategy are still unclear, and many patients may not be eligible due to significant comorbidities, previous HSCT, or lack of a suitable donor. Thus, the clinical utility of CD22-directed CAR T cells is limited by their inability to target malignant cells expressing low levels of antigen, similar to the experience of CD19 CAR T cells in lymphoma, and likely represents a fundamental problem for any therapy that targets antigens using T cells (or other immune effector cells) expressing second-generation CARs.

[0008] Thus, there is a need in the art for alternative approaches to generate engineered immune cells (e.g., T cells) that are useful as therapeutic agents. There is a need for new strategies to reduce relapse after CAR T cell therapy, improve patient outcomes by enhancing CAR T cell persistence and antigen sensitivity, and improve the clinical efficacy of CAR T cell therapy against various antigens and malignancies. The present invention addresses these unmet needs in the art. Summary of the Invention

[0009] The present disclosure provides a genetically modified immune cell comprising: a) a first chimeric antigen receptor (CAR) comprising an antigen recognition domain that binds to a first antigen, a transmembrane domain, and an intracellular signaling domain; and b) a second CAR comprising an antigen recognition domain that binds to the antigen, a transmembrane domain, and a linker for activation of T cells (LAT) intracellular signaling domain.

[0010] In some embodiments, the first antigen and the second antigen are different. In some embodiments, the first antigen and the second antigen are the same.

[0011] In some embodiments, the intracellular signaling domain of the first CAR comprises a CD3 zeta intracellular signaling domain. In some embodiments, the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:25, preferably, the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:24.

[0012] In some embodiments, the intracellular signaling domain of the first CAR further comprises at least one additional intracellular signaling domain selected from the group consisting of CD97 intracellular signaling domain, CD11a-CD18 intracellular signaling domain, CD2 intracellular signaling domain, ICOS intracellular signaling domain, CD27 intracellular signaling domain, CD154 intracellular signaling domain, CD8a intracellular signaling domain, OX40 intracellular signaling domain, 4-1BB intracellular signaling domain, CD28 intracellular signaling domain, ZAP40 intracellular signaling domain, CD30 intracellular signaling domain, GITR intracellular signaling domain, HVEM intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, MyD88 intracellular signaling domain, 2B4 intracellular signaling domain, and any combination thereof. In some embodiments, the at least one additional intracellular signaling domain is a 4-1BB intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 17.

[0013] In some embodiments, the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of any one of SEQ ID NOs: 26-34, and preferably, the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 27.

[0014] In some embodiments, the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 26, with a lysine to arginine substitution at position 25 of SEQ ID NO: 26 (K25R), a glycine to glutamic acid substitution at position 133 of SEQ ID NO: 26 (G133E), a lysine to arginine substitution at position 206 of SEQ ID NO: 26 (K206R), or any combination of the foregoing substitutions.

[0015] In some embodiments, the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 32, with a lysine to arginine substitution at position 25 of SEQ ID NO: 32 (K25R), a glycine to glutamic acid substitution at position 104 of SEQ ID NO: 32 (G104E), a lysine to arginine substitution at position 177 of SEQ ID NO: 32 (K177R), or any combination of the foregoing substitutions.

[0016] In some embodiments, the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 33, with a lysine to arginine substitution at position 25 of SEQ ID NO: 33 (K25R), a glycine to glutamic acid substitution at position 103 of SEQ ID NO: 33 (G103E), a lysine to arginine substitution at position 176 of SEQ ID NO: 33 (K176R), or any combination of the foregoing substitutions.

[0017] In some embodiments, the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 34, with a lysine to arginine substitution at position 25 of SEQ ID NO: 34 (K25R), a glycine to glutamic acid substitution at position 132 of SEQ ID NO: 34 (G132E), a lysine to arginine substitution at position 205 of SEQ ID NO: 34 (K205R), or any combination of the foregoing substitutions.

[0018] In some embodiments, the transmembrane domain of the first CAR and / or the second CAR comprises a transmembrane domain selected from the group consisting of CD8a transmembrane domain, CD28 transmembrane domain, CD3z transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, 2B4 transmembrane domain, BTLA transmembrane domain, and any combination thereof. In some embodiments, the transmembrane domain of the first CAR is derived from a CD8 alpha transmembrane domain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the transmembrane domain of the second CAR is derived from a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14.

[0019] In some aspects, the antigen recognition domain of the first CAR and / or the antigen recognition domain of the second CAR is an antibody, an antibody fragment, a single chain antibody, a single domain antibody, an scFv, a VH, or a VHH, or an antigen-binding fragment thereof.

[0020] In some embodiments, the antigen recognition domain of the first CAR and the antigen recognition domain of the second CAR further comprise a leader domain selected from the group consisting of: a CD8α leader domain, hi some embodiments, the leader domain is a CD8α leader domain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0021] In some embodiments, the first antigen and the second antigen are tumor associated antigens. In some embodiments, the tumor associated antigen is selected from the group consisting of CD19, CD22, CD20, CD138, BCMA, CD33, CD123, FLT, CLL, CD56, CD34, CD117, CD14, CD133, CD44v6, CD47, CD64, CD96, CD97, CD99, CD45, CD9, Muc1, Lewis-Y, IL1RAP, FR-beta, CD5, CD7, CD38, CD30, B7-H3, HER2, CD44v6, CEA, c-Met, EGFRvIII, Epcam, EphA2, FR-alpha, GD2, GPC3, IL13R-alpha2, IL11R-alpha, L1-CAM, mesothelin, MUC1, MUC16, NKGD2, and PSCA. In some embodiments, the first antigen is CD22. In some embodiments, the second antigen is CD19.

[0022] In some embodiments, the immune cells are T cells, natural killer (NK) cells, natural killer (NK)-like cells, cytokine-induced killer (CIK) cells, hematopoietic progenitor cells, peripheral blood (PB)-derived T cells, or umbilical cord blood (UCB)-derived T cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are iPS-derived immune cells.

[0023] In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 102, SEQ ID NO: 306, or SEQ ID NO: 309. In some embodiments, the second CAR comprises the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 100, SEQ ID NO: 206, or SEQ ID NOs: 300-308.

[0024] In some embodiments, the genetically modified immune cells comprise a first CAR comprising the amino acid sequence of SEQ ID NO: 102 and a second CAR comprising SEQ ID NO: 100. In some embodiments, the genetically modified immune cells comprise a first CAR comprising the amino acid sequence of SEQ ID NO: 102 and a second CAR comprising the amino acid sequence of SEQ ID NO: 306. In some embodiments, the genetically modified immune cells comprise a first CAR comprising the amino acid sequence of SEQ ID NO: 309 and a second CAR comprising the amino acid sequence of SEQ ID NO: 100.

[0025] The present disclosure provides a composition comprising a genetically modified immune cell of the present disclosure and a pharma- ceutically acceptable carrier.

[0026] The present disclosure provides compositions comprising a cell population, wherein a plurality of cells of the population comprises a genetically modified immune cell of the present disclosure. In some embodiments, the plurality of cells of the population comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage therebetween of a genetically modified immune cell of the present disclosure.

[0027] The present disclosure provides a polynucleotide encoding a first CAR and a second CAR of the present disclosure. In some aspects, a nucleic acid sequence encoding a self-cleaving peptide sequence is located between the nucleic acid sequence encoding the first CAR and the nucleic acid sequence encoding the second CAR. In some aspects, the self-cleaving peptide sequence comprises the amino acid sequence of SEQ ID NO: 79. In some aspects, the first CAR and the second CAR are encoded on a single vector. In some aspects, the vector is a viral vector, a lentiviral vector, a non-viral vector, or a transposon. In some aspects, the vector is a bicistronic lentiviral vector.

[0028] The present disclosure provides a method of generating a population of genetically modified immune cells, the method comprising: a) introducing a composition comprising a polynucleotide sequence of the present disclosure into a plurality of immune cells, thereby generating a population of genetically modified immune cells; b) culturing the population of genetically modified immune cells under conditions suitable for incorporation of the polynucleotide; and c) expanding and / or selecting at least one cell from the population of genetically modified immune cells that expresses a first CAR and a second CAR on the surface of the cell.

[0029] The present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering a composition of the present disclosure. In some embodiments, administration of a composition comprising engineered immune cells comprising a first CAR and a second CAR increases an immune response against the target cells compared to administration of a composition comprising engineered immune cells comprising only the first CAR. In some embodiments, the increase in immune response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage therebetween, greater than a composition comprising engineered immune cells comprising only the first CAR. In some embodiments, the cancer is a solid tumor, a B cell malignancy, a myeloid malignancy, a T cell malignancy, acute lymphoblastic leukemia, acute lymphoblastic lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelogenous leukemia, T lymphoblastic leukemia, T lymphoblastic lymphoma, or anaplastic large cell leukemia. In some embodiments, the cancer has low cell surface expression of the first antigen and / or low cell surface expression of the second antigen. [Brief description of the drawings]

[0030] [Figure 1A-D]Figures 1A-D show that antigen density influences CAR T cell efficacy and signaling through LAT. Figure 1A is an image showing NSG mice inoculated with NALM6 expressing no, low, or WT levels of CD22. Five days later, mice were treated with CD22 CART cells generated from healthy donors. Leukemia progression was followed by bioluminescence imaging. Figures 1B and 1C are western blots showing Jurkat cells stably expressing CD22 CAR stimulated for 2, 5, or 10 minutes with NALM6 cells expressing no, low, or WT levels of CD22. Western blot analysis was performed on lysates and probed for phosphate and total ZAP70 (Figure 1B) and LAT (Figure 1C). Figure 1D is a histogram showing CD22 CART cells co-incubated for 15 minutes with NALM6 cells expressing no, low, WT, or high levels of the CD22 antigen. Cells were fixed and permeabilized, and phospho-ERK was assessed by flow cytometry.

[0031] [Figure 2A-B] 2A-B show the design of exemplary bicistronic LAT-CAR and ALA-CAR constructs disclosed herein. Figure 2A is a schematic diagram of a standard second generation (Gen) (2G) CD22 CAR. Figure 2B is a schematic diagram of an exemplary bicistronic LAT-CAR or ALA-CAR that includes a first CAR (e.g., a 2G CD22 CAR) expressed with a second CAR (e.g., a "LAT-CAR" or "ALA-CAR", such as a CD19-directed CAR incorporating a LAT intracellular domain that amplifies the CAR response to low antigens).

[0032] [Figure 2C-F]Figures 2C-F show that bicistronic LAT-CAR increases the antigen sensitivity of CD22 CAR. Figure 2C is a bioluminescence image of the whole body of NSG mice inoculated with 106 low CD22 NALM6 and treated with 3x106 or 2.5x106 standard 2G CD22 CART (CD22 CART) cells or bicistronic LAT-CAR T cells (ALA-CART) or left untreated (NoTx), followed by twice weekly BLI. Figure 2D is a line graph showing quantification of the BLI imaging shown in Figure 2C. Figure 2E is a graph showing survival of the treated mouse cohort of Figure 2C. Figure 2F is a series of graphs showing analysis of bone marrow samples obtained from surviving mice treated with bicistronic LAT-CAR T cells of Figure 2C, showing continued persistence of bicistronic LAT-CAR T cells 50 days after initial treatment.

[0033] [Diagram 3] Figure 3 is a graph showing that 2G-CAR T cells reduced leukemia killing in vitro against low CD22 NALM6. CD22 2G-CAR T cells were generated from healthy donor T cells and co-incubated 1:1 E:T with GFP+NALM6 cells expressing WT (equivalent triangles) or low (inverted triangles) levels of CD22 antigen for 6 days. Leukemia cell killing was monitored over time by flow cytometry. Leukemia cell numbers were normalized to bead counts during co-culture and are shown on the y-axis. Days of co-culture are shown on the x-axis.

[0034] [Figure 4]Figure 4 is a series of flow cytometry histograms showing the enrichment of CAR-positive T cells after transduction. T cells from a healthy donor were activated and transduced with lentivirus containing a bicistronic CD22 / 19LAT-CAR construct. After 2 days, surface expression of CAR was determined by staining cells with fluorescently labeled CD22-Fc and CD19-Fc (top). CAR+ cells were positively selected using Miltenyi beads and T cells were expanded for an additional 4 days. At the end of expansion, T cells were stained again for surface CAR expression (bottom) and enrichment of CAR+ cells for downstream experiments is shown.

[0035] [Diagram 5] FIG. 5 is a series of graphs showing the surface co-expression of the first and second CARs of a bicistronic CAR of the present disclosure (top panel), as well as the relative intensity of surface expression of the first CAR of the present disclosure (ALA-CART-CD22BBz) compared to a standard second generation CAR (2G CD22BBz) as measured by flow cytometry (bottom panel).

[0036] [Figure 6] 6 is a series of graphs showing the surface expression of CAR constructs utilizing different transmembrane domains in the second CAR (e.g., LAT CAR) of the bicistronic CAR of the present disclosure. The use of the LAT transmembrane domain in the LAT-CAR resulted in minimal expression of the bicistronic CAR of the present disclosure on the surface of T cells from three healthy donors (top), while the incorporation of a transmembrane domain from the CD28 molecule into the second CAR (e.g., LAT CAR) of the bicistronic CAR construct disclosed herein resulted in efficient surface expression of the LAT CAR in T cells from the same healthy donor (bottom).

[0037] [Figure 7]Figure 7 is a series of Western blot images and graphs showing increased expression of LAT and increased activating phosphorylation of LAT (p-LAT225) in cells transduced with a bicistronic CAR construct of the present disclosure ("ALA-CART" or "22x19 ALACART") in response to normal (+) or low (Low) levels of CD22 on leukemia cells compared to cells transduced with 2G CD22Bz ("22Bz").

[0038] [Figure 8] Figure 8 is a series of Western blot images and graphs showing enhanced expression levels of total phospholipase C-gamma (PLCg) and activation of PLCg by phosphorylation (p-PLCg) in cells transduced with a bicistronic CAR construct of the present disclosure ("22x19LAT" or "22x19 ALACART") in response to normal (+) or low (Low) levels of CD22 on leukemia cells compared to cells transduced with 2G CD22Bz ("22Bz").

[0039] [Figure 9] FIG. 9 is a graph showing leukemia killing by CAR T cells as a ratio of leukemia cells versus CAR cells in cultures comprising NALM6 leukemia cells expressing various combinations of CD19 and CD22 antigens (DN double negative, 19-, 22-, WT, or low 22) and bicistronic CAR T cells of the disclosure (22×19LAT) or a CD22 CAR control.

[0040] [Figure 10] Figure 10 is a series of graphs showing hIL-2 and hIFNg concentrations (measured by ELISA) in cultures comprising NALM6 leukemia cells co-cultured with bicistronic CAR T cells (22x19LAT) of the present disclosure.

[0041] [Figure 11A]FIG. 11A is a series of images showing whole-body bioluminescence imaging (BLI) analysis in mice with leukemia expressing wild-type levels of CD22 and subsequently treated with a bicistronic CAR construct of the present disclosure (ALA-CART) compared to mice treated with a standard second generation CAR (CD22 CART) and mice that received no treatment (No Treatment).

[0042] [Figure 11B] FIG. 11B is a graph showing quantification of bioluminescence imaging (BLI) analysis in mice treated with the bicistronic CAR construct of the present disclosure (22×19 ALACART) or the second generation CAR construct (CD22BBzCAR) of FIG. 11A.

[0043] [Figure 11C] Figure 11C is a flow cytometry plot and graph showing analysis of bone marrow samples taken from mice treated with standard second generation CARs compared to mice treated with an exemplary bicistronic CAR construct of the present disclosure 50 days after CAR T cell infusion. These data demonstrate enhanced persistence of the bicistronic CAR T cells disclosed herein (ALA-CART) compared to standard second generation CAR T cells (CD22 CART).

[0044] [Figure 12A-12D]Figures 12A-12D are a series of charts, flow cytometry plots, and graphs showing the increased persistence in vivo of the bicistronic CAR of the present disclosure ("22x19LAT" or "22x19ALA-CART"). Figure 12A is a series of graphs showing flow cytometry analysis of bone marrow samples taken from mice treated with standard second generation CAR T cells ("22SA") and bone marrow samples taken from mice treated with the bicistronic CAR T cells of the present disclosure ("22x19LAT"). These data show that the enhanced persistence of the CAR T cells of the present disclosure ("22x19LAT") is primarily driven by the persistence of CD4+ CAR T cells (top panel) compared to CD8+ CAR T cells (bottom panel). Figure 12B is a series of flow cytometry histograms showing reduced expression of the exhaustion marker CD39 on the surface of the bicistronic CAR T cells of the present disclosure ("22x19ALACART") compared to standard second generation CD22 CART cells ("22BBz") 50 days after CAR T cell infusion. Figure 12C is a series of flow cytometry plots and summary graphs showing analysis of various T cell populations in samples from mice treated with bicistronic CAR T cells of the present disclosure ("22x19ALA-CART") and standard second generation CD22 CART cells ("22SA") 50 days after CAR T cell infusion. These data show an increased proportion of CAR T cells of the present disclosure with a central memory (CM) phenotype, which correlates with long-term persistence. Figure 12D is a series of flow cytometry plots, histograms, and summary graphs showing analysis of IL-7 receptor alpha (IL7RA) expression on CAR T cells obtained from mice treated with bicistronic CAR T cells of the present disclosure ("22x19 ALACART" or "22x19LAT") and standard second generation CAR22 CAR T cells ("22BBz").These results show that IL7RA expression is increased in CD4 T cells with effector memory (EM) and effector memory expressing CD45RA (T-EMRA) subpopulations in bicistronic CAR T cells and second generation CD CART cells, suggesting an enhanced capacity for long-term persistence of these cells.

[0045] [Figure 13A-13B] 13A-13B are a series of image data and graphs showing that an exemplary bicistronic LAT-CAR (ALA-CART) is effective against each targeted antigen. FIG. 13A is a series of images showing bioluminescence imaging (BLI) analysis in mice inoculated with leukemia expressing both antigens targeted by the bicistronic CAR construct of the present disclosure (WT NALM6CD19+ / CD22+) or expressing one or the other antigens targeted by the CAR of the present disclosure (CD19-NALM6(CD22+) or CD22-NALM6(CD19+)). Leukemia-bearing mice were treated with the bicistronic CAR T cells of the present disclosure (ALA-CART), standard second generation CAR T cells (CD22 CART), and left untreated (no treatment). Leukemia was eradicated by the bicistronic CAR T cells of the present disclosure, regardless of which antigen was present on the leukemia. Figure 13B is a graph showing the percentage of CAR T cells in bone marrow samples obtained from mice treated with bicistronic CAR T cells of the present disclosure after complete clearance of leukemia, demonstrating the persistence of bicistronic CAR T cells of the present disclosure in response to leukemia expressing both (WT) or either (CD19-, CD22-) of the target antigens.

[0046] [Figures 14A-14C]Figures 14A-14C are a series of flow cytometry histograms and graphs showing phosphorylation of signaling molecules in exemplary bicistronic CAR T cells (22x19LAT) or second generation CD22 CART cells (22BBz) of the present disclosure co-cultured with NALM6 leukemia cells expressing none (DN), both (WT) or one or the other (19-, 22-) of the targeted antigen. Figure 14A shows ERK (p-ERK) expression. Figure 14B shows p38 (p-p38) expression. Figure 14C shows PLCg (p-PLCg) expression.

[0047] [Figure 15] FIG. 15 shows images and graphs of quantified bioluminescence imaging (BLI) analysis in mice inoculated with low CD22 leukemia and treated with bicistronic CAR constructs of the present disclosure designed to target only the CD22 antigen (SAff / SAff-LAT, SAff / HiAff-LAT, HiAff / SAff-LAT, HiAff / HiAff-LAT) and mice treated with standard CD22 CART cells (22SAff (SEQ ID NO: 69)). Various combinations of antigen binding domains (scFvs) were tested utilizing standard affinity (SAff) and high affinity (HiAff) scFvs in the first, second, or both CARs of the constructs of the present disclosure. Among these various combinations, the use of high affinity scFvs in both CARs (HiAff / HiAff) showed the best elimination of low CD22 leukemia.

[0048] [Figure 16]Figure 16 shows images and graphs of quantified bioluminescence imaging (BLI) analysis in mice inoculated with leukemia expressing normal (NALM6WT) or low (NALM6 22low) levels of CD22 antigen and subsequently treated with a bicistronic CAR construct of the present disclosure utilizing high affinity scFvs at both positions ("HiAff / HiAff LAT" or "22ALACART4"), a standard second generation CD22 CAR (22SAff), and non-transduced T cells (mock). These data demonstrate the ability of the HiAff / HiAffLAT form of the present disclosure to eradicate low CD22 leukemia while targeting only the CD22 antigen.

[0049] [Figures 17A-17D]Figures 17A-17D show a series of graphs showing flow cytometry analysis of the phenotype of CAR cells of the present disclosure upon completion of manufacturing compared to the phenotype of standard second generation CD22 CAR T cells (22BBz). Various forms of the disclosure analyzed in this figure include CAR T cells targeting only CD22 with standard affinity scFvs on both CARs (22ALACART1), CAR T cells targeting only CD22 with a standard affinity scFv on the first CAR and a high affinity scFv on the second CAR (22ALACART2), CAR T cells targeting only CD22 with a high affinity scFv on the first CAR and a standard affinity scFv on the second CAR (22ALACART3), CAR T cells targeting only CD22 with high affinity scFvs on both CARs (22ALACART4), CAR T cells targeting CD22 and CD19 with a standard affinity CD22 scFv on the first CAR and a CD19-targeting scFv on the second CAR (22x19 ALACART). Phenotypic analysis of T cell subsets including T stem cell memory (Tscm), central memory (Tcm), effector memory (Tem), and effector memory re-expressing CD45RA (temra) was analyzed in CD4 (Figure 17A) and CD8 (Figure 17C) CAR T cells. IL-7 receptor alpha (IL7RA) surface expression was also assessed on CD4 (Figure 17B) and CD8 (Figure 17D) CAR T cells. These data indicate that transduction of T cells with the bicistronic CAR constructs of the present disclosure resulted in a CAR T cell product composed of a higher percentage of Tscm cells than standard second generation CARs, regardless of the scFv combination used. Similarly, IL7RA expression was uniformly higher in all configurations of bicistronic CAR T cells disclosed herein compared to IL7RA expression in standard CD22 CAR cells.

[0050] [Figure 18]FIG. 18 is a series of graphs showing flow cytometry analysis of the expression of the marker CD39 associated with T cell exhaustion on T cells transduced with various configurations of the bicistronic CAR (22-ALA-CART) of the present disclosure (SAff / SAff-LAT, SAff(SA) / HiAff-LAT, HiAff / SAff(SA)-LAT, HiAff / HiAff-LAT) and on T cells transduced with a standard second generation CD22 CAR T (22SA). The analysis of T cells was subdivided into the analysis of CD4+ CAR ("CAR4") (top) and CD8+ CAR ("CAR8") (bottom) CAR T cells. Expression of the CD39 exhaustion marker was lower on T cells transduced with any of the configurations of the bicistronic CAR disclosed herein than on T cells transduced with a standard second generation CD22 CAR.

[0051] [Figure 19] Figure 19 is a series of whole-body bioluminescence images showing leukemia progression and in vivo activity of exemplary bicistronic LAT-CAR T cells (19ALA-CART) in mice compared to standard second generation CD19 CAR T cells (CD19BBz) and non-transduced T cell (sham) controls in mice. Images were taken from day 1 (D-1) to day 14 (D14) after T cell injection, as indicated. Bioluminescence activity is indicated by color (radiance).

[0052] [Figure 20] Figure 20 is a series of whole-body bioluminescence images showing leukemia progression and in vivo efficacy of exemplary bicistronic LAT-CAR T cells (19ALA-CART) in mice transplanted with high CD19 NALM6 cells compared to standard second generation CD19 CAR T cells (CD19BBz) and non-transduced T cell (sham) controls. Images were taken from day 1 (D-1) to day 42 (D42) after T cell injection, as indicated. Bioluminescence activity is indicated by color (radiance).

[0053] [Figure 21]Figure 21 is a graph of CAR T cell-mediated killing of low CD22 leukemia cells after overnight co-culture with exemplary bicistronic 22ALA-CART cell variants (LAT-WT (SEQ ID NO:26), LAT-K52R (SEQ ID NO:27), LAT-233R (SEQ ID NO:28), LAT-K52R+K233R (SEQ ID NO:29)) compared to control T cells (sham) at multiple ratios. The ratio of effector CAR T cells to target leukemia cells (E:T ratio) is shown on the x-axis. Cell killing is shown as specific lysis (%) on the y-axis.

[0054] [Fig. 22A-22B] Figures 22A-22B are graphs of CAR T cell-mediated killing of low CD22 leukemia cells after overnight co-culture with exemplary 22ALA-CART variants (LAT-K52R (SEQ ID NO:27), LAT-K52R+G160E (SEQ ID NO:30), LAT-K52R+K233R (SEQ ID NO:29), LAT-K52R+K233R+G160E (SEQ ID NO:31)) at multiple ratios compared to control T cells (sham). The ratio of effector CAR T cells to target leukemia cells (E:T ratio) is shown on the x-axis. Cell killing is shown as specific lysis (%) on the y-axis. Figure 22A shows cell killing by LAT-CAR with mutations at ubiquitination site K52 (LAT-K52R+G160E, LAT-K52R+K233R+G160E) or LAT-CAR without the PLC-activating mutation G160E (LAT-K52R). Figure 22B shows cell killing by LAT-CAR with mutations at ubiquitination sites K52 and K233 (LAT-K52R+G160E) or LAT-CAR without the PLC-activating mutation G160E (LAT-K52R+K233R).

[0055] [Figure 23A-23B]Figures 23A-23B are a series of graphs showing the function of bicistronic LAT-CAR T cells (ALA-CART) compared to standard second generation CD22 CART cells. Figure 23A is a graph of quantification of cytokines IL-2 and interferon gamma (IFNg) produced by either bicistronic ALA-CART cells (22x19 ALACART) or standard second generation CD22 CART cells (22BBz) after overnight co-culture with low CD22 NALM6 cells or CD22(-) NALM6 cells. Figure 23B is a graph showing specific lysis of low CD22 NALM6 cells and CD22(-) NALM6 cells by either bicistronic ALA-CART cells (22x19 ALACART) or standard second generation CD22 CART cells (22BBz) after overnight co-culture at various E:T ratios. **** indicates statistical significance with a p-value of <0.0001.

[0056] [Figures 24A-24C] Figures 24A-24C show a series of whole-body bioluminescence images and graphs showing the in vivo persistence of a CAR of the present disclosure that targets NALM6 via recognition of the CD22 antigen alone. Figure 24A shows bioluminescence images of mice engrafted with WT NALM6 leukemia and treated with bicistronic LAT-CAR T cells of the present disclosure that target only CD22 (22ALA-CART), mice treated with standard second generation CD22 CART cells (22BBz), and mice treated with non-transduced (mock) T cells. Figure 24B is a series of graphs showing quantification of persistent bicistronic CAR T cells (22ALACART4) or second generation CD22 CART cells (22BBz) in the bone marrow of mice 40 days after initial treatment, showing enhanced in vivo persistence of the bicistronic CAR of the present disclosure (22ALACART4). Figure 24C is a series of graphs showing quantification of the differentiation state (CM, EM, and TEMRA) of the persistent bicistronic CAR T cells and second generation CD22 CART cells of Figure 24A, showing an increase in the percentage of CARs of the present disclosure with a memory phenotype.

[0057] [Fig. 25A-25B] Figures 25A-25B are a series of graphs showing the phenotype of exemplary CAR cells of the present disclosure at the completion of manufacturing compared to standard CD22 CART cells (22BBz). Figure 25A is a series of pie charts showing phenotypic analysis of T cell subsets including T stem cell memory (TSCM), central memory (TCM), effector memory, and effector memory re-expressing CD45RA (TEMRA) in bicistronic CAR T cells of the present disclosure (22ALA-CART) compared to standard second generation CAR T cells (22BBz). Figure 25B is a series of graphs showing the percentage of T cells with TSCM phenotype (CD4+CAR ("CAR4") or CD8+CAR ("CAR8")) from three different T cell donors after manufacturing of the CAR of the present disclosure (22ALA-CART) and standard second generation CAR (22BBz). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] The invention generally provides cells (including immune cells (e.g., T cells, B cells, natural killer (NK) cells, monocytes, macrophages, or artificially generated cells with immune effector function) from patients, healthy donors, differentiated stem cells (including, but not limited to, induced pluripotent stem cells (iPSCs), embryonic stem cells, hematopoietic stem cells and / or other tissue-specific stem cells), or non-human sources) that have been genetically engineered to express a first antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) that binds one antigen, along with a second antigen-recognizing receptor (e.g., a CAR) that comprises an intracellular signaling domain of a linker for T cell activation (LAT) that binds a second antigen, and methods of use thereof for the treatment of cancer, infectious diseases, autoimmunity, alloimmunity, lymphoproliferative disorders, pathological immune dysregulation, and other conditions in which an increased antigen-specific immune response is desired or to facilitate transplantation of solid organs or hematopoietic stem cells. The first and second CARs may recognize the same or different epitopes on the same antigen, or epitopes found on two different antigens. Activation of immune cells (e.g., T cells) is mediated by engagement of the first CAR to its cognate antigen (e.g., CD22) or engagement of the second CAR containing the LAT intracellular domain to its cognate antigen (e.g., CD19), with signal amplification resulting in enhanced persistence, antigen sensitivity, and efficacy that occurs when both the first and second CARs simultaneously engage their respective cognate antigens (e.g., CD22 and CD19).

[0059] CARs, sometimes also referred to as artificial T cell receptors, chimeric T cell receptors (cTCRs), T bodies, or chimeric immune receptors, are engineered receptors that are currently well known in the art. They are primarily used to transform immune effector cells, particularly T cells, to provide them with the desired antigen specificity and effector response. Adoptive cell therapy using CAR-T cells is particularly under investigation in the field of cancer treatment. In these therapies, T cells are harvested from the patient, donor, or derived from stem cell sources and engineered to express a CAR specific for an antigen found in a particular form of cancer. When CAR-T cells, which can recognize and kill cancer cells, are reintroduced into the patient, they proliferate, eliminate target antigen-positive cells, and in a small number of patients, transition into a long-lasting population while retaining anti-tumor effector activity.

[0060] First-generation CARs provide TCR-like signals from immunoreceptor tyrosine-based activation motifs (ITAMs), most commonly containing intracellular signaling domains derived from CD3 zeta (CD3z) molecules, thereby exerting tumoricidal functions. However, engagement of CD3z chain fusion receptors may not be sufficient to cause substantial IL-2 secretion and / or T cell proliferation in the absence of concurrent costimulatory signals. In physiological T cell responses, optimal lymphocyte activation requires engagement of one or more costimulatory receptors, such as CD28 or 4-1BB. In the context of suboptimal activation produced by first-generation CARs, T cell activity in vivo is often transient and fails to control malignancies.

[0061] Second generation CARs have been constructed to deliver functional antigen-dependent costimulatory signals in human primary T cells in addition to antigen-dependent TCR-like signals, allowing T cell proliferation in addition to tumoricidal activity. Second generation CARs most commonly use costimulatory domains (synonymous costimulatory signaling regions) derived from CD28 or 4-1BB to provide costimulation. The combined delivery of costimulation and CD3 zeta signals makes second generation CARs superior in terms of function compared to their first generation counterparts (CD3z signals only). One example of a second generation CAR is described in U.S. Patent No. 7,446,190, which is incorporated herein by reference.

[0062] Third generation CARs have also been prepared. These combine multiple costimulatory domains (synonymously, costimulatory signaling regions) in cis with a TCR-like signaling domain, such as CD28+4-1BB+CD3z or CD28+OX40+CD3z, to further increase potency. In third generation CARs, the costimulatory domains are arranged in tandem in the CAR endodomain, generally located upstream of CD3z or its equivalent. In general, however, the results achieved with these third generation CARs have been disappointing, showing only slight improvements over second generation constructs, with some third generation CARs being inferior to second generation constructs.

[0063] The present invention is the first to utilize a first CAR (i.e., a first, second, or third generation CAR) in combination with a second CAR with an intracellular signaling domain of LAT as a means to amplify CAR signaling and increase persistence and antigen sensitivity. Unlike the first CAR, the second CAR lacks a TCR-like signaling region such as CD3z. These T cells engineered to express a dual CAR system show superior activity and persistence compared to first, second, and third generation CAR-T cells. Thus, the present invention overcomes the challenges associated with current technology by providing antigen-specific immune cells (e.g., T cells) for immunotherapy, such as the treatment of immune-related diseases, including cancer, autoimmune diseases, and infectious diseases.

[0064] The present invention is based at least in part on the discovery that low levels of antigen reduce the utilization of linker for T cell activation (LAT) downstream of CAR. LAT is a scaffolding protein that functions as a key component of the signalosome and has been shown to amplify the signal generated by antigen receptors in T cells by increasing cytokine release after receptor activation. The introduction of a second LAT-containing chimeric antigen receptor induces significantly higher levels of LAT activation upon antigen stimulation than the second generation CAR itself.

[0065] The present invention is based at least in part on the discovery that the simultaneous engagement of two antigens co-expressed by tumor cells by a first costimulatory ITAM-containing receptor and a second LAT-containing antigen-recognition receptor is useful for activating and stimulating immune-reactive cells.In particular, the reactivity to cells expressing only one of the antigens may be reduced compared to the reactivity to cells expressing both antigens due to lack of coordinated signaling, but productive T cell activation can also occur against target cells expressing low levels of either target antigen.However, the activation of T cells in the presence of both antigens is greater than the activation of T cells by only one of the CARs.Thus, this approach increases the reactivity of T cells against tumors expressing low levels of tumor-associated antigens.

[0066] The sensitivity of CARs to their cognate antigens has a significant impact on the outcome of patients undergoing CAR T therapy. We generated multiple subclones of the precursor B ALL cell line NALM6 that express various amounts of CD22 antigen, and found that CAR T cell cytokine production, cytotoxicity, effector differentiation, persistence, and in vivo efficacy are significantly reduced when levels of CD22 fall below 1500-2000 molecules per cell. The effect of antigen density on CAR T cell function is not unique to CD22 CAR cells, as CAR T cells directed against CD19, CD20, HER2, ALK, and B7-H3 have all been shown to have reduced activity against low antigen targets. Furthermore, recent clinical observations have associated low levels of CD19 antigen with treatment failure and / or relapse in patients undergoing CD19-directed CAR T cell therapy for diffuse large cell B cell lymphoma.

[0067] The effect of low antigen sensitivity of CAR T cells has been described, but the underlying mechanism remains to be elucidated. High sensitivity to low levels of antigen is a characteristic of conventional T cells activated through endogenous T cell receptors (TCRs), with evidence that T cell activation occurs in response to less than 10 antigen-MHC complexes / cell, and full effector responses occur in response to less than 200 antigen-MHC complexes / cell. TCR sensitivity is due in part to the formation of a highly organized immune synapse and the subsequent formation of a signalosome around an assembly of LAT molecules, where the T cell's signaling machinery localizes to the antigen binding site and amplifies proximal signaling events to activate multiple branched downstream signaling pathways. In contrast, CARs do not form a well-organized immune synapse to aggregate the necessary components of the signalosome to the intracellular receptor activation site. Disorganization of the CAR immune synapse and subsequent inefficient assembly and utilization of the signalosome leads to suboptimal signaling within T cells, impairing T cell responses to low levels of antigen and compromising higher level T cell function, such as the establishment of long-lived, persistent CAR T cell populations in vivo.

[0068] The inability of CAR T cells to target low levels of antigens is of immediate clinical importance, as this is the primary mechanism of relapse in patients treated with CD22 CAR T cells, the most proven treatment option for patients with CD19-negative leukemia after immunotherapy. Similarly, there is growing evidence that low levels of CD19 antigen are associated with an increased risk of initial treatment failure and relapse in patients with diffuse large cell B-cell lymphoma. Clinical studies of B-cell maturation antigen (BCMA)-directed CAR T cells suggest that the initial efficacy of CAR T cells is reduced in patients with multiple myeloma who express low levels of the targeted BCMA antigen. Furthermore, reduced expression of BCMA is commonly observed during disease progression and / or relapse after CAR T cell therapy, further highlighting the clinical importance of enabling CAR T cells to efficiently target low antigen malignant cells.

[0069] Thus, the present invention provides a novel approach to address the shortcomings of current CAR T cell therapy by improving the ability of T cells to recognize tumor cells expressing low levels of antigens and increasing the persistence of CAR T cells, thereby improving clinical patient outcomes.

[0070] The immune cells of the present disclosure may be targeted to any combination of antigens, and exemplary antigens for the CARs disclosed herein include, but are not limited to, CD22 and CD19. In certain aspects, the immune cells are targeted to any combination of antigens, including CD19 and CD20, CD20 and CD22, CD19 and CD79a, CD22 and CD79a, CD20 and CD79a, CD19 and CD79b, CD22 and CD79b, CD20 and CD79b, CD19 and CD5, CD138 and BCMA, CD38 and BCMA, CD19 and BCMA, CD19 and CD138, CD19 and GPRC5D, BCMA and GPRC5D, CD138 and GPRC5D, CD38 and GPRC5D, CD5 and CD7, CD19 and CD20 and CD22. D5 and TCR alpha or beta chain, CD7 and TCR alpha or beta chain, CD5 and CD38, CD7 and CD38, CD30 and ALK, CD33 and FLT3, CD33 and CD123, CD33 and CLEC1A, CD33 and CD56, CD33 and CD34, CD33 and CD117, CD33 and CD14, CD33 and CD133, CD33 and CD44v6, CD33 and CD47, CD33 and CD64, CD33 and CD96, CD33 and CD97, CD33 and CD99 , CD33 and CD16, CD33 and CD45, CD33 and CD9, CD33 and Muc1, CD33 and Lewis-Y, CD33 and IL1-RAP, CD33 and FR-beta, CD33 and ROR1, CD123 and FLT3, CD123 and CLEC1A, CD123 and CD56, CD123 and CD34, CD123 and CD117, CD123 and CD14, CD123 and CD133, CD123 and CD44v6, CD123 and CD47, CD123 and CD64, CD123 and CD96, CD123 and CD97, CD123 and CD99, CD123 and CD16, CD123 and CD45, CD123 and CD9, CD123 and Muc1, CD123 and Lewis-Y, CD123 and IL1-RAP, CD123 and FR-beta, CD123 and ROR1, FLT3 and CLEC1A, FLT3 and CD56, FLT3 and CD34, FLT3 and CD117, FLT3 and CD14, FLT3 and CD133, FLT3 and CD44v6, FLT3 and CD47,FLT3 and CD64, FLT3 and CD96, FLT3 and CD97, FLT3 and CD99, FLT3 and CD16, FLT3 and CD45, FLT3 and CD9, FLT3 and Muc1, FLT3 and Lewis-Y, FLT3 and IL1-RAP, FLT3 and FR-beta, FLT3 and ROR1, CLEC1A and CD56, CLEC1A and CD34, CLEC1A and CD117, CLEC1A and CD14, CLEC1A and CD133, CLEC1A and CD44v6, CLEC1A and CD47, CLEC1A and CD64, C LEC1A and CD96, CLEC1A and CD97, CLEC1A and CD99, CLEC1A and CD16, CLEC1A and CD45, CLEC1A and CD9, CLEC1A and Muc1, CLEC1A and Lewis-Y, CLEC1A and IL1-RAP, CLEC1A and FR-beta, CLEC1A and ROR1, CD56 and CD34, CD56 and CD117, CD56 and CD14, CD56 and CD133, CD56 and CD44v6, CD56 and CD47, CD56 and CD64, CD56 and CD96, CD56 and CD 97, CD56 and CD99, CD56 and CD16, CD56 and CD45, CD56 and CD9, CD56 and Muc1, CD56 and Lewis-Y, CD56 and IL1-RAP, CD56 and FR-beta, CD56 and ROR1, CD34 and CD117, CD34 and CD14, CD34 and CD133, CD34 and CD44v6, CD34 and CD47, CD34 and CD64, CD34 and CD96, CD34 and CD97, CD34 and CD99, CD34 and CD16, CD34 and CD45, CD34 and CD9, CD34 and Muc1, CD34 and Lewis-Y, CD34 and IL1-RAP, CD34 and FR-beta, CD34 and ROR1, CD117 and CD14, CD117 and CD133, CD117 and CD44v6, CD117 and CD47, CD117 and CD64, CD117 and CD96, CD117 and CD97, CD117 and CD99, CD117 and CD16, CD117 and CD45, CD117 and CD9, CD117 and Muc1, CD117 and Lewis-Y, CD117 and IL1-RAP, CD117 and FR-beta,CD117 and ROR1, CD14 and CD133, CD14 and CD44v6, CD14 and CD47, CD14 and CD64, CD14 and CD96, CD14 and CD97, CD14 and CD99, CD14 and CD16, CD14 and CD45, CD14 and CD9, CD14 and Muc1, CD14 and Lewis-Y, CD14 and IL1-RAP, CD14 and FR-beta, CD14 and ROR1, CD133 and CD44v6, CD133 and CD47, CD133 and CD64, CD133 and CD96, CD133 and CD97 , CD133 and CD99, CD133 and CD16, CD133 and CD45, CD133 and CD9, CD133 and Muc1, CD133 and Lewis-Y, CD133 and IL1-RAP, CD133 and FR-beta, CD133 and ROR1, CD44V6 and CD47, CD44V6 and CD64, CD44V6 and CD96, CD44V6 and CD97, CD44V6 and CD99, CD44V6 and CD16, CD44V6 and CD45, CD44V6 and CD9, CD44V6 and Muc1, CD44V6 and Lewis-Y, CD44V6 and IL1-RAP, CD44V6 and FR-beta, CD44V6 and ROR1, CD47 and CD64, CD47 and CD96, CD47 and CD97, CD47 and CD99, CD47 and CD16, CD47 and CD45, CD47 and CD9, CD47 and Muc1, CD47 and Lewis-Y, CD47 and IL1-RAP, CD47 and FR-beta, CD47 and ROR1, CD64 and CD96, CD64 and CD97, CD64 and CD99, CD64 and CD16, CD64 and CD45, CD64 and CD9, CD64 and Muc1, CD64 and Lewis-Y, CD64 and IL1-RAP, CD64 and FR-beta, CD64 and ROR1, CD96 and CD97, CD96 and CD99, CD96 and CD16, CD96 and CD45, CD96 and CD9, CD96 and Muc1, CD96 and Lewis-Y, CD96 and IL1-RAP, CD96 and FR-beta, CD96 and ROR1, CD97 and CD99, CD97 and CD16, CD97 and CD45, CD97 and CD9, CD97 and Muc1, CD97 and Lewis-Y,CD97 and IL1-RAP, CD97 and FR-beta, CD97 and ROR1, CD99 and CD16, CD99 and CD45, CD99 and CD9, CD99 and Muc1, CD99 and Lewis-Y, CD99 and IL1-RAP, CD99 and FR-beta, CD99 and ROR1, CD16 and CD45, CD16 and CD9, CD16 and Muc1, CD16 and Lewis-Y, CD16 and IL1-RAP, CD16 and FR-beta, CD16 and ROR1, CD45 and CD9, CD45 and Muc1, CD45 and Lewis s-Y, CD45 and IL1-RAP, CD45 and FR-beta, CD45 and ROR1, CD9 and Muc1, CD9 and Lewis-Y, CD9 and IL1-RAP, CD9 and FR-beta, CD9 and ROR1, MUC1 and Lewis-Y, MUC1 and IL1-RAP, MUC1 and FR-beta, MUC1 and ROR1, Lewis-Y and IL1-RAP, Lewis-Y and FR-beta, Lewis-Y and ROR1, IL1-RAP and FR-beta, IL1-RAP and ROR1, FR-beta and ROR1, B7-H3 and and HER2, B7-H3 and CD44v6, B7-H3 and CEA, B7-H3 and CD133, B7-H3 and c-Met, B7-H3 and EGFRvIII, B7-H3 and EPCAM, B7-H3 and EPHA2, B7-H3 and FR-alpha, B7-H3 and GD2, B7-H3 and GPC3, B7-H3 and IL-13R-alpha2, B7-H3 and IL-11R-alpha, B7-H3 and L1-CAM, B7-H3 and mesothelin, B7-H3 and MUC1, B7-H3 and MUC16, B7-H3 and IL1-RAP, B7-H3 and CD99, B7-H3 and PSCA, B7-H3 and PSMA, B7-H3 and ROR1, B7-H3 and ALK, HER2 and CD44v6, HER2 and CEA, HER2 and CD133, HER2 and c-Met, HER2 and EGFRvIII, HER2 and EPCAM, HER2 and EPHA2, HER2 and FR-alpha, HER2 and GD2, HER2 and GPC3, HER2 and IL-13R-alpha2, HER2 and IL-11R-alpha, HER2 and L1-CAM, HER2 and mesothelin, HER2 and MUC1,HER2 and MUC16, HER2 and IL1-RAP, HER2 and CD99, HER2 and PSCA, HER2 and PSMA, HER2 and ROR1, HER2 and ALK, CD44v6 and CEA, CD44v6 and CD133, CD44v6 and c-Met, CD44v6 and EGFRvIII, CD44v6 and EPCAM, CD44v6 and EPHA2, CD44v6 and FR-alpha, CD44v6 and GD2, CD44v6 and GPC3, CD44v6 and IL-13R-alpha2, CD44v6 and IL-11R-alpha, CD44v6 and L1-CAM, CD44v6 and mesothelin, CD44v6 and MUC1, CD44v6 and MUC16, CD44v6 and IL1-RAP, CD44v6 and CD99, CD44v6 and PSCA, CD44v6 and PSMA, CD44v6 and ROR1, CD44v6 and ALK, CEA and CD133, CEA and c-Met, CEA and EGFRvIII, CEA and EPCAM, CEA and EPHA2, CEA and FR-alpha, CEA and GD2, CEA and GPC3, CEA and IL-13R-alpha2, CEA and and IL-11R-alpha, CEA and L1-CAM, CEA and mesothelin, CEA and MUC1, CEA and MUC16, CEA and IL1-RAP, CEA and CD99, CEA and PSCA, CEA and PSMA, CEA and ROR1, CEA and ALK, CD133 and c-Met, CD133 and EGFRvIII, CD133 and EPCAM, CD133 and EPHA2, CD133 and FR-alpha, CD133 and GD2, CD133 and GPC3, CD133 and IL-13R-alpha2, CD133 and IL-11R-alk pha, CD133 and L1-CAM, CD133 and mesothelin, CD133 and MUC1, CD133 and MUC16, CD133 and IL1-RAP, CD133 and CD99, CD133 and PSCA, CD133 and PSMA, CD133 and ROR1, CD133 and ALK, c-Met and EGFRvIII, c-Met and EPCAM, c-Met and EPHA2, c-Met and FR-alpha, c-Met and GD2, c-Met and GPC3, c-Met and IL-13R-alpha2, c-Met and IL-11R-alpha,c-Met and L1-CAM, c-Met and mesothelin, c-Met and MUC1, c-Met and MUC16, c-Met and IL1-RAP, c-Met and CD99, c-Met and PSCA, c-Met and PSMA, c-Met and ROR1, c-Met and ALK, EGFRvIII and EPCAM, EGFRvIII and EPHA, 2, EGFRvIII and FR-alpha, EGFRvIII and GD2, EGFRvIII and GPC3, EGFRvIII and IL-13R-alpha2, EGFRvIII and IL-11R-alpha, EGFRvIII and L1-CAM, EGFRvIII and mesothelin, EGFRvIII and MUC1, EGFRvIII and MUC16, EGFRvIII and IL1-RAP, EGFRvIII and CD99, EGFRvIII and PSCA, EGFRvIII and PSMA, EGFRvIII and ROR1, EGFRvIII and ALK , EPCAM and EPHA2, EPCAM and FR-alpha, EPCAM and GD2, EPCAM and GPC3, EPCAM and IL-13R-alpha2, EPCAM and IL-11R-alpha, EPCAM and L1-CAM, EPCAM and mesothelin, EPCAM and MUC1, EPCAM and MUC16, EPCAM and IL1-RAP, EPCAM and CD99, EPCAM and PSCA, EPCAM and PSMA, EPCAM and ROR1, EPCAM and ALK, EPHA2 and FR-alpha, EPHA2 and GD2, EPHA2 and and GPC3, EPHA2 and IL-13R-alpha2, EPHA2 and IL-11R-alpha, EPHA2 and L1-CAM, EPHA2 and mesothelin, EPHA2 and MUC1, EPHA2 and MUC16, EPHA2 and IL1-RAP, EPHA2 and CD99, EPHA2 and PSCA, EPHA2 and PSMA, EPHA2 and ROR1, EPHA2 and ALK, FR-alpha and GD2, FR-alpha and GPC3, FR-alpha and IL-13R-alpha2, FR-alpha and IL-11R-alpha, FR-alpha and and L1-CAM, FR-alpha and mesothelin, FR-alpha and MUC1, FR-alpha and MUC16, FR-alpha and IL1-RAP, FR-alpha and CD99, FR-alpha and PSCA, FR-alpha and PSMA, FR-alpha and ROR1, FR-alpha and ALK, GD2 and GPC3, GD2 and IL-13R-alpha2, GD2 and IL-11R-alpha, GD2 and L1-CAM, GD2 and mesothelin, GD2 and MUC1, GD2 and MUC16, GD2 and IL1-RAP, GD2 and CD99,GD2 and PSCA, GD2 and PSMA, GD2 and ROR1, GD2 and ALK, GPC3 and IL-13R-alpha2, GPC3 and IL-11R-alpha, GPC3 and L1-CAM, GPC3 and mesothelin, GPC3 and MUC1, GPC3 and MUC16, GPC3 and IL1-RAP, GPC3 and CD99, GPC3 and PSCA, GPC3 and PSMA, GPC3 and ROR1, GPC3 and ALK, IL-13R-alpha2 and IL-11R-alpha, IL-13R-alpha2 and L1-CAM, IL-13R-alpha IL-13R-alpha2 and mesothelin, IL-13R-alpha2 and MUC1, IL-13R-alpha2 and MUC16, IL-13R-alpha2 and IL1-RAP, IL-13R-alpha2 and CD99, IL-13R-alpha2 and PSCA, IL-13R-alpha2 and PSMA, IL-13R-alpha2 and ROR1, IL-13R-alpha2 and ALK, IL-11R-alpha and L1-CAM, IL-11R-alpha and mesothelin, IL-11R-alpha and MUC1, IL-11R-alpha and MUC16, IL-1 1R-alpha and IL1-RAP, IL-11R-alpha and CD99, IL-11R-alpha and PSCA, IL-11R-alpha and PSMA, IL-11R-alpha and ROR1, IL-11R-alpha and ALK, L1-CAM and mesothelin, L1-CAM and MUC1, L1-CAM and MUC16, L1-CAM and IL1-RAP, L1-CAM and CD99, L1-CAM and PSCA, L1-CAM and PSMA, L1-CAM and ROR1, L1-CAM and ALK, mesothelin and MUC1, mesothelin and M UC16, mesothelin and IL1-RAP, mesothelin and CD99, mesothelin and PSCA, mesothelin and PSMA, mesothelin and ROR1, mesothelin and ALK, MUC1 and MUC16, MUC1 and IL1-RAP, MUC1 and CD99, MUC1 and PSCA, MUC1 and PSMA, MUC1 and ROR1, MUC1 and ALK, MUC16 and IL1-RAP, MUC16 and CD99, MUC16 and PSCA, MUC16 and PSMA, MUC16 and ROR1, MUC16 and ALK, IL1-RAP and CD99,Dual targeting against combinations of antigens including, but not limited to, IL1-RAP and PSCA, IL1-RAP and PSMA, IL1-RAP and ROR1, IL1-RAP and ALK, CD99 and PSCA, CD99 and PSMA, CD99 and ROR1, CD99 and ALK, PSCA and PSMA, PSCA and ROR1, PSCA and ALK, PSMA and ROR1, PSMA and ALK, ROR1 and ALK. In any of the foregoing antigen combinations, either the first CAR or the second CAR (e.g., the first costimulatory and ITAM-containing CAR and the second LAT-containing antigen-recognizing CAR) can be specific for any of the combined antigens. In a non-limiting example, in the combination of CD20 and CD22 antigens, the first CAR (costimulatory and ITAM-containing CAR) can be specific for CD20 and the second CAR (LAT-containing antigen-recognizing CAR) can be specific for CD22, or the first CAR (costimulatory and ITAM-containing CAR) can be specific for CD22 and the second CAR (LAT-containing antigen-recognizing CAR) can be specific for CD20.

[0071] Furthermore, expression of two CARs increases the specificity of the T cells by limiting off-target toxicity of the cells, such that a signal is provided to the T cell to kill only if the cell comes into contact with both antigens expressed on the tumor, resulting in enhanced proliferation and persistence in vivo. Thus, normal cells that express only one antigen may not be targeted by the T cells of the present disclosure.

[0072] Genetic reprogramming of immune cells, such as NK cells and T cells, for adoptive cancer immunotherapy has clinically relevant applications and advantages, such as 1) increased ability to recognize tumor cells expressing low levels of antigens, and 2) increased cell persistence and proliferation. Thus, the present disclosure also provides methods for treating immune-related disorders, such as cancer, including adoptive cellular immunotherapy using any of the engineered immune cells provided herein.

[0073] I. Definition As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the particular components are intentionally incorporated into the composition and / or are present only as contaminants or in trace amounts.Thus, the total amount of the particular component resulting from any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%.Most preferred is a composition in which the particular component is not detectable using standard analytical methods.

[0074] As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0075] As used herein, the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, but the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" may mean at least two or more.

[0076] As used herein, the term "about" is used to indicate that a value includes the inherent error variation of the device, the method used to determine the value, or the variation that exists among study subjects.

[0077] As used herein, the term "portion," when used in reference to a polypeptide or peptide, refers to a fragment of the polypeptide or peptide. In some embodiments, a "portion" of a polypeptide or peptide retains at least one function and / or activity of the full-length polypeptide or peptide from which it is derived. For example, in some embodiments, if a full-length polypeptide binds to a given ligand, then a portion of the full-length polypeptide also binds to the same ligand.

[0078] The terms "protein" and "polypeptide" are used interchangeably herein.

[0079] The term "exogenous" when used with respect to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means, or with respect to a cell, the term refers to a cell that has been isolated and then introduced into a cell population or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or may be one or more additional copies of a nucleic acid that naturally occurs in an organism or cell. An exogenous cell may be from a different organism or may be from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid that is at a chromosomal location different from where it occurs in a natural cell, or a nucleic acid that is adjacent to a nucleic acid sequence that is different from the nucleic acid sequence found in nature. The term "exogenous" is used interchangeably with the term "heterologous".

[0080] "Expression construct" or "expression cassette" is used to mean a nucleic acid molecule capable of directing transcription. An expression construct contains at least one or more transcription control elements (such as promoters, enhancers, or functional equivalents thereof) that direct gene expression in one or more desired cell types, tissues, or organs. Additional elements, such as transcription termination signals, may also be included.

[0081] A "vector" or "construct" (sometimes referred to as a gene delivery system or gene transfer "vehicle") refers to a macromolecule or molecular complex comprising a polynucleotide, or a protein expressed by the polynucleotide, that is delivered to a host cell in vitro or in vivo.

[0082] A "plasmid", a common type of vector, is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA. In some cases, it is circular and double-stranded.

[0083] An "origin of replication" ("ori") or "replication origin" is a DNA sequence that, when present in a plasmid in a cell, is capable of maintaining the plasmid and / or linked sequences at or near the site where DNA synthesis is initiated. As an example, the ori of EBV (Epstein-Barr Virus) contains FR sequences (20 imperfect copies of a 30 bp repeat) and preferably DS sequences. However, other sites in EBV bind EBNA-1, e.g., Rep * The FR, DS, or Rep sequences can serve as an alternative to the DS as a replication origin (Kirshmaier and Sugden, 1998). Thus, the EBV replication origin does not contain the FR, DS, or Rep sequences. * The sequences include any functionally equivalent sequences or synthetic combinations derived therefrom by nucleic acid modification. For example, the methods of the present disclosure may also use an EBV origin of replication that has been genetically engineered, such as by insertion or mutation of individual elements.

[0084] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgene" "encoding" a particular protein is a portion of a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed in vitro or in vivo and, optionally, translated into a gene product, e.g., a polypeptide. The coding region may be present in either cDNA, genomic DNA, or RNA form. If present in DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Genes include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence is typically located 3' to the gene sequence.

[0085] The term "control elements" refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, splice junctions, and the like, which collectively provide for the replication, transcription, post-transcriptional processing, and translation of a coding sequence in a recipient cell. Not all of these control elements need be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

[0086] The term "promoter" is used herein to refer to a nucleotide region that includes DNA regulatory sequences, derived from a gene, that are capable of binding RNA polymerase and initiating transcription of a downstream protein (3' direction) coding region. It may include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, bind to initiate specific transcription of a nucleic acid sequence. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence to control transcription initiation and / or expression of that sequence.

[0087] By "enhancer" is meant a nucleic acid sequence that, when placed adjacent to a promoter, results in increased transcriptional activity compared to the transcriptional activity resulting from the promoter in the absence of the enhancer domain.

[0088] "Operably linked" with respect to nucleic acid molecules means that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and a functional effector element) are linked in a manner that allows for transcription of the nucleic acid molecule. "Operably linked" with respect to peptide and / or polypeptide molecules means that two or more peptide and / or polypeptide molecules are linked in a manner that results in a single polypeptide chain having at least one property of each peptide and / or polypeptide component of the fusion, i.e., a fusion polypeptide. Fusion polypeptides are preferably chimeric, i.e., composed of molecules not naturally found in a single polypeptide.

[0089] The term "homology" refers to the percent identity between the nucleic acid residues of two polynucleotides or the amino acid residues of two polypeptides. The correspondence between one sequence and another can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information between two polypeptides by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of the polynucleotides under conditions that promote the formation of stable duplexes between the homologous regions, followed by digestion with a single-strand specific nuclease, and sizing of the digested fragments. Two polynucleotide (e.g., DNA) or two polypeptide sequences are "substantially homologous" to each other if at least about 80%, at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids match, respectively, over a defined length of the molecule, as determined using the methods described above.

[0090] The term "cell" is used herein in the broadest sense in the art and refers to a structural unit of tissue in a multicellular organism, surrounded by a membrane structure that isolates the cell from the outside, capable of self-replication, and having genetic information and a mechanism for expressing it. As used herein, a cell may be a naturally occurring cell or an artificially modified cell (e.g., a fused cell, a genetically modified cell, etc.).

[0091] The term "stem cell" as used herein refers to a cell that, under suitable conditions, can differentiate into a diverse range of specialized cell types, but under other suitable conditions, can self-renew and remain in an essentially undifferentiated pluripotent state. The term "stem cell" also encompasses pluripotent cells, multipotent cells, precursor cells, and progenitor cells. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be generated from somatic cells by reprogramming the somatic cells to a pluripotent state through the expression of certain transcription factors associated with pluripotency, and these cells are referred to as "induced pluripotent stem cells" or "iPSc", "iPSC", or "iPS cells".

[0092] "Embryonic stem (ES) cells" refer to undifferentiated pluripotent cells obtained from earlier stage embryos, such as the inner cell mass at the blastocyst stage, or generated by artificial means (e.g., nuclear transfer), and capable of giving rise to any differentiated cell type, including germ cells (e.g., sperm and eggs) in the embryo or adult.

[0093] "Induced pluripotent stem cells" (iPScs, iPSCs, or iPS cells) are cells generated by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPS cells can be generated using fetal, infant, neonatal, pediatric, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, Klf4, Nanog, and Lin28. In some embodiments, to reprogram somatic cells into pluripotent stem cells, the somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, at least four reprogramming factors, at least five reprogramming factors, at least six reprogramming factors, or at least seven reprogramming factors.

[0094] "Hematopoietic progenitor cells" or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic lineage but capable of further hematopoietic differentiation, including hematopoietic stem cells, multipotent hematopoietic stem cells, common myeloid progenitors, megakaryocyte progenitors, erythroid progenitors, and lymphoid progenitors. Hematopoietic stem cells (HSCs) are multipotent stem cells that generate all blood cell types, including myeloid (monocytes and macrophages, granulocytes (neutrophils, basophils, eosinophils, and mast cells), erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid (T cells, B cells, NK cells) lineages (see, e.g., Doulatov et al., 2012; Notta et al., 2015).

[0095] "Multiple lymphoid progenitors" (MLPs) are defined to represent any progenitor cell that generates all lymphoid lineages (B, T, and NK cells) but may or may not have other (myeloid) potential (Doulatov et al., 2010), CD45RA + / CD10 + / CD7 +Any B, T, and NK progenitor cell may be referred to as an MLP. "Common myeloid progenitor cells" (CMPs) are CD45RA + / CD135 + / CD10 + / CD7 + Refers to cells.

[0096] "Pluripotent stem cells" refer to stem cells that have the potential to differentiate into all of the cells that make up one or more tissues or organs, or preferably any of the three germ layers: endoderm (stomach lining, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), or ectoderm (epidermal tissue and nervous system).

[0097] The term "somatic cell" as used herein refers to any cell other than a germ cell, such as an egg, sperm, etc., that does not directly transmit its DNA to the next generation. Typically, somatic cells have limited or no pluripotency. As used herein, somatic cells may be naturally occurring or genetically modified.

[0098] "Programming" is the process of changing the type of progeny that a cell can produce. For example, a cell is programmed if it is modified to be able to form at least one new cell type progeny in culture or in vivo compared to what it would have been able to form under the same conditions without programming. This essentially means that if no such progeny could be formed before programming, after sufficient proliferation, a measurable proportion of progeny having the phenotypic characteristics of the new cell type is observed, or the proportion having the characteristics of the new cell type is clearly greater than before programming. This process includes differentiation, dedifferentiation, and transdifferentiation.

[0099] "Differentiation" is the process by which a less specialized cell becomes a more specialized cell type. "Dedifferentiation" is the cellular process by which a partially or terminally differentiated cell reverts to an earlier developmental stage, such as pluripotency or multipotency. "Transdifferentiation" is the process by which one differentiated cell type is converted to another differentiated cell type. Typically, transdifferentiation by programming occurs without the cell passing through an intermediate pluripotency stage, i.e., the cell is directly programmed from one differentiated cell type to another differentiated cell type. Under certain conditions, the proportion of progeny with characteristics of the new cell type can be at least about 1%, 5%, 25%, or more, in order of increasing priority.

[0100] As used herein, the term "subject" or "subject in need thereof" refers to a mammal, male or female, of any age, preferably a human, in need of therapeutic intervention, cell transplantation, or tissue transplantation. Typically, the subject is in need of therapeutic intervention, cell transplantation, or tissue transplantation (also referred to herein as the recipient) due to a disorder, pathological or undesirable condition, state, or syndrome, or a physical, morphological, or physiological abnormality amenable to therapeutic intervention, cell transplantation, or tissue transplantation.

[0101] As used herein, "disruption" or "alteration" in reference to a gene refers to a homologous recombination event in a nucleic acid molecule (e.g., an endogenous gene sequence) that results in the elimination or reduction of expression of one or more gene products encoded by the gene of interest in a cell, compared to the expression level of the gene product in the absence of the disruption. Exemplary gene products include the mRNA and protein products encoded by the gene of interest. The alteration is in some cases transient or reversible, and in other cases permanent. The alteration is in some cases an alteration of a functional or full-length protein or mRNA, despite the fact that truncated or non-functional products may be generated. In some embodiments herein, the activity or function of a gene is disrupted, as opposed to expression. Genetic alteration is generally induced by artificial methods, i.e., the addition or introduction of a compound, molecule, complex, or composition, and / or by alteration of the nucleic acid of the gene or associated with the gene, such as at the DNA level. Exemplary methods for genetic alteration include genetic alteration techniques, such as gene silencing, knockdown, knockout, and / or gene editing. Examples of gene editing methods include CRISPR / Cas systems, meganuclease systems, zinc finger protein (ZFP) and zinc finger nuclease (ZFN) systems, and / or transcription activator-like proteins (TAL), transcription activator-like effector proteins (TALE), or TALE nuclease proteins (TALEN) systems. Examples of genetic alterations also include antisense technologies such as RNAi, siRNA, shRNA, and / or ribozymes, which generally result in a transient reduction in expression, and gene editing technologies that result in targeted gene inactivation or alteration, such as by induction of cleavage and / or homologous recombination. Examples include insertions, mutations, and deletions. Alterations typically result in the suppression and / or complete absence of expression of the normal or "wild-type" product encoded by the gene. Examples of such genetic alterations are insertions, frameshift and missense mutations, deletions, substitutions, knock-ins, and knock-outs of genes or parts of genes, including deletion of the entire gene.Such alterations can occur in the coding region, e.g., one or more exons, resulting in the inability to produce a full-length product, a functional product, or any product, such as by inserting a stop codon. Such alterations can also occur by altering promoters or enhancers, or other regions that affect transcriptional activation and prevent transcription of the gene. Gene alterations include gene targeting, including inactivation of targeted genes by homologous recombination.

[0102] "Immune disorder," "immune-related disorder," or "immune-mediated disorder" refers to a disorder in which the immune response plays a significant role in the development or progression of the disease. Immune-mediated disorders include autoimmune diseases, allograft rejection, graft-versus-host disease, and inflammatory and allergic conditions.

[0103] An "immune response" is the response of a cell of the immune system, such as a NK cell, a B cell, or a T cell, or an innate immune cell, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response").

[0104] As used herein, the term "antigen" is a molecule that can be bound by an antibody, a T cell receptor, a chimeric antigen receptor, and / or an engineered immune receptor. Antigens may generally be used to induce a humoral and / or cellular immune response that results in the production of B and / or T lymphocytes.

[0105] The terms "tumor-associated antigen," "tumor antigen," and "cancer cell antigen" are used interchangeably herein. In each case, the term refers to a protein, glycoprotein, or carbohydrate that is specifically or preferentially expressed by cancer cells.

[0106] An "epitope" is a site on an antigen that is recognized by an antibody, determined by the specificity of the amino acid sequence. Two antibodies are said to bind to the same epitope if each competitively inhibits (prevents) the binding of the other to the antigen, as measured in a competitive binding assay. Alternatively, two antibodies bind to the same epitope if most amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies are said to have overlapping epitopes if each partially inhibits binding to the antigen of the other and / or if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0107] "Autoimmune disease" refers to a disease 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., self-antigens), resulting in tissue damage. Self-antigens may be derived from host cells or from commensal organisms, such as microorganisms that normally colonize mucosal surfaces (known as commensals).

[0108] The term "graft-versus-host disease (GVHD)" refers to a common serious complication of bone marrow or other tissue transplants in which a reaction of donated immunocompetent lymphocytes occurs against the transplant recipient's own tissue. GVHD is a possible complication of any transplant that uses or includes stem cells from related or unrelated donors. In some embodiments, the GVHD is chronic GVHD (cGVHD).

[0109] A "parameter of immune response" is any specific measurable aspect of an immune response, including, but not limited to, cytokine secretion (such as IFN-γ), chemokine secretion, changes in migration or cell accumulation, immunoglobulin production, maturation of dendritic cells, regulatory activity, immune cell numbers, and proliferation of any cell of the immune system. Another parameter of an immune response is structural damage or functional impairment of any organ resulting from an immunological attack. One skilled in the art can easily determine an increase in any one of these parameters using known laboratory assays. In one specific, non-limiting example, to assess cell proliferation: 3 H-thymidine incorporation can be evaluated. A "substantial" increase in a parameter of immune response is a significant increase in this parameter compared to a control. Specific non-limiting examples of a substantial increase are at least about 50% increase, at least about 75% increase, at least about 90% increase, at least about 100% increase, at least about 200% increase, at least about 300% increase, and at least about 500% increase. Similarly, an inhibition or reduction in a parameter of immune response is a significant decrease in this parameter compared to a control. Specific non-limiting examples of a substantial decrease are at least about 50% decrease, at least about 75% decrease, at least about 90% decrease, at least about 100% decrease, at least about 200% decrease, at least about 300% decrease, and at least about 500% decrease. Statistical tests such as non-parametric ANOVA and T-tests can be used to compare the difference in the magnitude of the response caused by the first agent compared to the percentage of samples that respond using the second agent. In some instances, p≦0.05 is significant, indicating that there is less than a 5% chance that any observed increase or decrease in a parameter is due to random variation. Those of skill in the art can readily identify other statistical assays to use.

[0110] "Treating" a disease or condition or treatment thereof refers to carrying out a protocol or treatment regimen that may include administering one or more drugs to a patient to alleviate the signs or symptoms of the disease or the recurrence of the disease. Desirable effects of treatment include slowing the rate of disease progression, improving or mitigating the disease state, and remission, increasing survival time, improving quality of life, or improving prognosis. Alleviation or prevention can occur before and after the signs or symptoms of the disease and condition appear. Thus, "treating" or "treatment" can also include "preventing" or the "prevention" of a disease or undesirable condition. Furthermore, "treating" or "treatment" does not require complete alleviation of signs or symptoms, nor does it require a cure, and specifically includes protocols or treatment regimens that have only a minor effect on the patient.

[0111] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to 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, reducing the frequency of signs or symptoms of a disease or their severity. For example, treating cancer may include, for example, reducing tumor size, reducing tumor invasiveness, reducing the rate of cancer growth, or preventing metastasis or recurrence. Treating cancer may also mean extending the survival of a subject with cancer.

[0112] "Antigen recognition moiety" or "antigen recognition domain" refers to a molecule or a portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, an antibody-like molecule, or a fragment thereof, and the antigen is a tumor antigen.

[0113] As used herein, "antibody" refers to a monoclonal or polyclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B cells and binding to the same epitope. In contrast, a "polyclonal antibody" refers to a population of antibodies produced by different B cells and binding to different epitopes of the same antigen. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CHL, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have a similar general structure, and each region contains four framework regions whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2 and CDR3, form the "hypervariable region" of an antibody that is responsible for antigen binding.

[0114] An "antibody-like molecule" may be, for example, a protein that is a member of the Ig superfamily that is capable of selectively binding to a partner.

[0115] The terms "fragment of an antibody," "antibody fragment," "functional fragment of an antibody," and "antigen-binding portion" are used interchangeably herein to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al. (2005) Nat. Biotech. 23(9):1126-29). An antibody fragment desirably contains, for example, one or more CDRs, a variable region (or portion thereof), a constant region (or portion thereof), or a combination thereof.

[0116] Examples of antibody fragments include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the stalk region; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of the two domains (i.e., VL and VH) of an Fv fragment joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al. (1988), Science 242:423-6; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83; and Osbourn et al. (1989) J. Immunol. 1999:1311-1323; (see, e.g., U.S. Pat. No. 6,331,363; see ...

[0117] "Chimeric antigen receptors" are also known as artificial cellular receptors, chimeric cellular receptors, or chimeric immune receptors. Chimeric antigen receptors (CARs) are engineered receptors that confer selected specificity to immune effector cells. CARs typically have an extracellular domain (extracellular domain) that contains an antigen-binding domain and a stalk region, a transmembrane domain, and an intracellular (endodomain) domain.

[0118] "Stalk region" encompasses the terms "spacer region" or "hinge domain" or "hinge" and is used to link an antigen binding domain to a transmembrane domain. As used herein, the term "stalk region" generally refers to any oligonucleotide or polypeptide that functions to link a transmembrane domain to either the extracellular or cytoplasmic domain in the polypeptide chain of a CAR. In embodiments, it has sufficient flexibility to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.

[0119] The term "functional portion," when used in reference to a CAR, refers to any portion or fragment of a CAR described herein, where the portion or fragment retains the biological activity of the CAR of which it is a part (the parent CAR). With respect to a nucleic acid sequence encoding a parent CAR, a nucleic acid sequence encoding a functional portion of a CAR can encode a protein that comprises, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0120] The term "functional variant" as used herein refers to a polypeptide or protein that has substantial or significant sequence identity or similarity with a reference polypeptide and retains the biological activity of the reference polypeptide of which it is a variant. Functional variants include, for example, variants of a CAR (parent CAR) described herein that retain the ability to recognize a target cell to a similar extent, to the same extent, or to a greater extent than the parent CAR. With respect to the nucleic acid sequence encoding the parent CAR, the nucleic acid sequence encoding the functional variant of the CAR can be, for example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, or about 99% identical to the nucleic acid sequence encoding the parent CAR.

[0121] The phrase "pharmacologically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other undesirable reactions when administered to an animal, such as a human, as appropriate. It will be understood that for animal (e.g., human) administration, preparations must meet sterility, pyrogenicity, general safety, and purity standards, e.g., as required by the FDA Office of Biological Standards.

[0122] As used herein, "pharmaceutical acceptable carriers" include any aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, liquids, and nutritional supplements, such like materials, and combinations thereof, known to those skilled in the art. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0123] The term "T cells" refers to T lymphocytes, including gamma / delta T cells, alpha / beta T cells, NK T cells, and CD4 + T cells and CD8 + These include, but are not limited to, CD4 + T cells include THO, T h 1, and TH2 cells, as well as regulatory T cells (T reg There are at least three types of regulatory T cells: CD4 + CD25 + T reg , CD25T H 3T reg , and CD25 TR1 T reg "Cytotoxic T cells" refer to T cells that can kill other cells. The majority of cytotoxic T cells are CD8+ Although MHC class I-restricted T cells, some cytotoxic T cells are CD4 + In a preferred embodiment, the T cells of the present disclosure are CD4 + or CD8 + It is.

[0124] The activation state of T cells can be determined by whether the T cells are "resting" (i.e., in the G phase of the cell cycle) and ready to proliferate following an appropriate stimulus, such as recognition of a specific antigen, or stimulation with an antibody, such as OKT3, PHA, or PMA. 0 The "phenotype" of a T cell (e.g., naive, central memory, effector memory, lytic effector, help effector (THI and TH2 cells), and regulatory effector) describes the function the cell will perform when activated. Healthy donors have T cells of each of these phenotypes that are primarily in a resting state. A naive T cell will proliferate upon activation and then differentiate into a memory T cell or an effector T cell. It can then become resting again and change phenotype again until it is next activated to perform a new function. An effector T cell will divide upon activation and antigen-specific effector function.

[0125] "Natural killer T cells" (NKT cells) (not to be confused with natural killer cells of the innate immune system) act as a bridge between the adaptive and innate immune systems. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (WIC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d. Once activated, these cells are able to exert functions attributed to both Th and Tc cells (i.e., production of cytokines and release of cytolytic / cell-killing molecules). They can also recognize and eliminate some tumor cells and cells infected with herpes viruses.

[0126] "Natural killer cells" ("NK cells") are a type of cytotoxic lymphocyte of the innate immune system. In some cases, NK cells provide the first line of defense against viral infection and / or tumor formation. NK cells detect MHC present on infected or cancer cells, triggering cytokine release and subsequent induction of lysis and apoptosis. NK cells can also detect stressed cells in the absence of antibody and / or MHC, allowing for a rapid immune response.

[0127] As used herein, "tumor antigen" refers to any antigenic substance that is produced, expressed, or overexpressed in a tumor cell, which may, for example, elicit an immune response in the host.

[0128] Alternatively, for purposes of this disclosure, tumor antigens may be proteins expressed by both healthy and tumor cells, but which identify a particular tumor type and are therefore suitable therapeutic targets. In one embodiment, the tumor antigen is CD22. In one embodiment, the tumor antigen is CD19.

[0129] The term "antigen-presenting cell (APC)" refers to a class of cells that can present one or more antigens in the form of peptide-MHC complexes that can be recognized by specific effector cells of the immune system, thereby inducing an effective cellular immune response against the presented antigens. APCs can be intact whole cells, such as macrophages, B cells, endothelial cells, activated T cells, and dendritic cells, or other naturally occurring or synthetic molecules, such as purified MHC class I molecules complexed with 2-microglobulin.

[0130] The term "culture" refers to the in vitro maintenance, differentiation, and / or proliferation of cells in a suitable medium. "Enriched" refers to a composition comprising cells that are present in a greater proportion (total cells) than the cells are found in the tissue in which they reside in an organism.

[0131] An "anti-cancer agent" can adversely affect a subject's cancer cells / tumor by, for example, promoting the death of cancer cells, inducing apoptosis in cancer cells, slowing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or prolonging the lifespan of a patient with cancer.

[0132] II. Immune cells Certain embodiments of the present disclosure relate to immune cells expressing chimeric antigen receptors (CARs). The immune cells include T cells (e.g., regulatory T cells, CD4 + T cells, CD8 + The cells may be inflammatory cells, such as inflammatory bowel disease (IGD) cells, immune ...

[0133] Immune cells may be isolated from a subject, particularly a human subject. Immune cells may be obtained from a subject of interest, 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 may be enriched or purified from any tissue in which immune cells are present, including, but not limited to, blood (including blood collected by a blood bank or umbilical cord blood bank), spleen, bone marrow, tissues removed and / or exposed during a surgical procedure, and tissues obtained by a biopsy procedure. The tissues / organs from which immune cells are enriched, isolated, and / or purified may be isolated from both living and non-living subjects, with the non-living subject being an organ donor. The isolated immune cells may be used directly or may be stored for a period of time, such as by freezing. In some embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some embodiments, immune cells isolated from umbilical cord blood have enhanced immunomodulatory capabilities, for example as measured by CD4+ or CD8+ T cell suppression. In certain embodiments, to enhance immune regulatory capacity, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood. Pooled blood may be from two or more sources, for example, 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

[0134] The population of immune cells can be obtained from a subject in need of therapy or from a subject suffering from a disease associated with reduced immune cell activity. Thus, the cells are autologous to the subject in need of therapy. Alternatively, the population of immune cells can be obtained from a donor. The immune cell population can be taken from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present in the subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, such as from pooled umbilical cord blood. The population of immune cells can be derived from induced pluripotent stem cells (iPSCs) and / or any other stem cells known in the art. In some aspects, the iPSCs and / or stem cells used to obtain the population of immune cells can be obtained from a subject in need of therapy or from a subject suffering from a disease associated with reduced immune cell activity, thus, these iPSCs and / or stem cells are autologous to the subject in need of treatment. Alternatively, the iPSCs and / or stem cells can be obtained from a donor, thus, allogeneic to the subject in need of treatment.

[0135] When the population of immune cells is obtained from a donor different from the subject, it is preferred that the donor is allogeneic, so long as the obtained cells are compatible with the subject in that they can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible. To match with the subject, allogeneic cells can be treated to reduce immunogenicity.

[0136] 1.T cells

[0137] T cells play a major role in cell-mediated immunity (antibodies are not involved). Their T cell receptor (TCR) distinguishes them from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem memory T cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, like naive cells, but express high amounts of CD95, IL-2R, CXCR3, and LFA-1, making them memory cells). These include: (i) central memory TCM cells, which express L-selectin and CCR7 and secrete IL-2 but not IFNg or IL-4; (ii) effector memory TCM cells, however, do not express L-selectin or CCR7 but produce effector cytokines such as IFNg and IL-4); regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells); natural killer T cells (NKT); and gamma delta T cells.

[0138] T cells for immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. Additionally, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a blood unit drawn from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0139] 2. Engineered antigen receptors

[0140] Immune cells of the present disclosure (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4 + T cells, CD8 + T cells, or gamma delta T cells), NK cells, invariant NK cells, NKT cells, stem cells (e.g., MSCs or iPS cells) can be genetically engineered to express an antigen receptor, such as an engineered CAR and / or TCR. For example, host cells (e.g., autologous or allogeneic T cells) are modified to express a CAR with antigen specificity for a cancer antigen. In certain embodiments, T cells are engineered to express a CAR. T cells may further be engineered to express a TCR. Multiple CARs and / or TCRs, such as for different antigens, may be added to a single cell type, such as a T cell.

[0141] Suitable modification methods are known in the art. For example, see Sambrook and Ausubel, supra. For example, cells can be transduced to express TCR with antigen specificity for cancer antigen using the transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009.

[0142] In some embodiments, the cells contain one or more nucleic acids introduced by genetic engineering that encode one or more antigen receptors, and the genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous. In some embodiments, the nucleic acids are non-naturally occurring, such as nucleic acids not found in nature (e.g., chimeras).

[0143] In some embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen (e.g., a tumor antigen or a pathogen antigen). In some embodiments, the antigen is a protein expressed on the surface of a cell (e.g., a cancer cell).

[0144] Exemplary engineered antigen receptors, including CARs and recombinant TCRs, and methods for engineering and introducing receptors into cells are described, for example, in WO2000 / 14257, WO2013126726, WO2012 / 129514, WO2014 / 031687, WO2013 / 166321, WO2013 / 071154, and WO2013 / 123061, U.S. Patent Application Publication Nos. US2002 / 131960, US2013 / 287748, and US2013 / 0149337, and U.S. Patent No. 6,451,999,626, and U.S. Pat. Nos. 5, 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, 7,446,190, 7,446,191, 8,324,353, and 8,479,118; those described in International Patent Application Publication No. WO 2014 / 055668 A1, and European Patent Application Publication No. EP 2 537 416, and / or Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012.

[0145] 3. Chimeric antigen receptor

[0146] In some aspects, the disclosure provides a population of genetically modified immune cells (e.g., T cells) engineered to express a first chimeric antigen receptor (CAR) and / or a polynucleotide encoding a CAR, where the CAR comprises (a) an antigen recognition domain that specifically binds a first antigen (e.g., CD22), a transmembrane domain, and an intracellular signaling domain, and (b) a second chimeric antigen receptor (CAR) and / or a polynucleotide encoding a CAR, where the second CAR comprises (a) an antigen recognition domain that specifically binds an antigen (which may be different from the antigen to which the first CAR binds (e.g., CD22 and CD19) or may be the same as the antigen to which the first CAR binds (e.g., CD22 and CD22), a transmembrane domain, and a LAT intracellular signaling domain. In some embodiments, the intracellular domain of the first CAR comprises one or more (e.g., one, two, three, or more) costimulatory domains.

[0147] In some embodiments, the engineered cells contain additional CARs, including activating or stimulatory CARs, costimulatory CARs (see, e.g., PCT Publication No. WO2014 / 055668), and / or inhibitory CARs (iCARs, see, e.g., Fedorov et al., 2013). CARs generally comprise an extracellular antigen (or ligand) recognition domain linked, in some aspects via a linker and / or a transmembrane domain, to one or more intracellular signaling components. Such molecules typically mimic or approximate signals through natural antigen receptors, signals through such receptors in combination with costimulatory receptors, and / or signals through costimulatory receptors alone. For example, when an antigen is recognized by the extracellular antigen recognition domain, the intracellular signaling components transmit an activation signal to the T cell that induces the T cell to destroy the targeted tumor cell.

[0148] A. Antigen Recognition Domain In some embodiments, the antigen recognition domain of the CAR described herein may recognize an epitope that includes a shared space between one or more antigens. In some embodiments, the antigen recognition domain comprises a complementarity determining region (CDR) of a monoclonal antibody, a variable region of a monoclonal antibody, a scFv, a VH, a VHH, a single domain antibody (e.g., a camelid single domain antibody), an antibody mimic, and / or an antigen-binding fragment thereof. In some embodiments, the specificity of the antigen recognition domain is derived from a protein or peptide (e.g., a ligand in a receptor-ligand pair) that specifically binds to another protein or peptide (e.g., a receptor in a receptor-ligand pair). In some embodiments, the antigen recognition domain comprises an aptamer, a T cell receptor (TCR)-like antibody, or a single chain TCR (scTCR). Almost any moiety that binds to a given target (e.g., a tumor-associated antigen (TAA)) with sufficient affinity can be used as an antigen recognition domain. The configuration of the antigen recognition domain can be multimeric, such as a diabody or multimer. In some embodiments, multimers can be formed by cross-pairing of variable portions of light and heavy chains into diabodies.

[0149] In some embodiments, the antigen recognition domain of the CAR described herein comprises an antibody mimic. The term "antibody mimic" is intended to refer to an organic compound that specifically binds to a target sequence and has a structure different from that of a naturally occurring antibody. An antibody mimic may include a protein, a nucleic acid, or a small molecule. The target sequence to which the antibody mimic of the present disclosure specifically binds may be an antigen. Exemplary antibody mimics include, but are not limited to, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers (also known as avidity multimers), DARPins (designed ankyrin repeat proteins), finomers, Kunitz domain peptides, monobodies, and sentinels.

[0150] In some embodiments, the first CAR provided herein comprises a single chain variable fragment (scFv) derived from a monoclonal antibody specific for a tumor-associated antigen (e.g., CD22), a hinge domain, a transmembrane domain, and an ITAM-containing intracellular signaling domain (e.g., CD3ζ). Such a molecule provides for the transduction of a signal via the ITAM in response to target recognition by the scFv. In some embodiments, the first CAR further comprises an additional intracellular signaling domain ("costimulatory domain").

[0151] In some embodiments, the second CAR provided herein comprises a single chain variable fragment (scFv) derived from a monoclonal antibody specific for a tumor-associated antigen (e.g., CD19), a hinge domain, a transmembrane domain, and a LAT intracellular signaling domain. Such a molecule provides for transduction of the LAT signal in response to target recognition by the scFv, amplifying the signal from the first CAR.

[0152] Nucleic acids encoding any of the CARs described herein are also provided. The nucleic acids encoding CARs may be humanized. In some embodiments, the nucleic acids encoding CARs provided herein are codon-optimized for expression in human cells. In some embodiments, the present disclosure provides full-length CAR cDNAs or coding regions.

[0153] In some embodiments, the antigen recognition domain of the CARs provided herein comprises a fragment of the VH and VL chains of a single chain variable fragment (scFv) that specifically binds to CD22. Thus, the antigen recognition domain of the CARs provided herein can comprise any scFv known in the art to specifically bind to CD22.

[0154] In some embodiments, the antigen recognition domain of the CARs provided herein comprises fragments of the VH and VL chains of a single chain variable fragment (scFv) that specifically binds to CD19, such as those described in U.S. Patent Application Publication No. 2020 / 0246382, PCT Application Publication No. WO2020223445, and PCT Application Publication No. WO2020123691, each of which is incorporated herein by reference in its entirety. Thus, the antigen recognition domain of the CARs provided herein can comprise any scFv known in the art to specifically bind to CD19.

[0155] In some embodiments, the antigen recognition domain of a CAR described herein binds (e.g., specifically binds) to an antigen set forth in Table 1. An antigen-specific CAR, when expressed on the cell surface, redirects the specificity of an immune cell (e.g., a T cell) to the respective antigen.

[0156] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17]

[0157] In some embodiments, the antigen recognition domain of a CAR described herein binds (e.g., specifically binds) to at least one of L1-CAM, mesothelin, MUC1, MUC16, NKGD2, PSCA, PSMA, ROR1, and ALK. Antigen-specific CARs, when expressed on the cell surface, redirect the specificity of immune cells (e.g., T cells) to their respective antigens.

[0158] In some embodiments, the antigen recognition domain of a CAR described herein binds (e.g., specifically binds) CD22. A CD22-specific CAR, when expressed on the cell surface, redirects the specificity of a T cell to human CD22 (see, e.g., Accession Nos. NM_001178098; NM_001770; NM_001385732 and NP_001171569; NP_001761).

[0159] In some embodiments, the antigen recognition domain of a CAR described herein binds (e.g., specifically binds) CD19. A CD19-specific CAR, when expressed on the cell surface, redirects the specificity of a T cell to human CD19 (e.g., accession numbers: NM_001178098; NM_001770; NM_001385732 and NP_001171569; NP_001761).

[0160] i) an antigen recognition domain comprising an anti-CD22 antibody or a fragment thereof In some embodiments, the antigen recognition domain of the CARs provided herein comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen recognition domain of the CARs provided herein comprises a single-chain antibody fragment (scFv) comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) of a monoclonal anti-CD22 antibody. Optionally, the VH and VL may be linked by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In some embodiments, the antigen-binding portion may comprise a VH and VL directionally linked, for example, from N-terminus to C-terminus (VH-linker-VL or VL-linker-VH).

[0161] In some embodiments, the antigen recognition domain of the CARs provided herein comprises an scFv that is affinity optimized for CD22 to induce cytotoxicity of tumor cells that produce high or normal levels of CD22. In some embodiments, the antigen recognition domain of the CARs provided herein comprises an scFv that is affinity optimized for CD22 to induce cytotoxicity of tumor cells that produce low levels of CD22.

[0162] Exemplary anti-CD22 scFvs from which the antigen recognition domain for use in the CARs described herein may be derived include, but are not limited to, m971 and immunologically active and / or antigen-binding fragments thereof. Thus, in some embodiments, the antigen recognition domain of the CARs provided herein comprises a VH and a VL derived from any one of the anti-CD22 antibodies m971. In some embodiments, the antigen recognition domain of the CARs provided herein comprises a VH and a VL separated by a linker.

[0163] The amino acid sequences of the VH (and corresponding CDRH1, CDRH2, and CDRH3) and VL (and corresponding CDRL1, CDRL2, and CDRL3) of high affinity m971 and low affinity m971 are provided below. The affinity of "standard affinity" m971 is approximately KD=3.1 nM. The affinity of "high affinity" m971 is approximately KD=18 pM (Ramakrishna et al, Clin Cancer Res, 2019. PMID:31110075).

[0164] High affinity m971 full length amino acid sequence: [ka] High affinity m971-VH-amino acids: [ka] High affinity m971-VL-amino acids: [ka] High affinity m971 linker: GGGGSGGGSGGGGS (SEQ ID NO: 211) High affinity M971-CDRH1: GDSVSSNSVA (SEQ ID NO: 212) High affinity M971-CDRH2: TYYRSTWYN (SEQ ID NO: 213) High affinity M971-CDRH3:AREVTGDLEDAFDI (SEQ ID NO:86) High affinity M971-CDRL1: QTIWSY (SEQ ID NO: 87) High affinity M971-CDRL2:AAS (SEQ ID NO:88) High affinity M971-CDRL3: QQSYSIPQT (SEQ ID NO:89) High affinity m971 full length nucleic acid: [ka] Standard affinity m971 full length-amino acid [ka] Standard affinity m971 linker: GGGGS (SEQ ID NO:215) Standard affinity m971scFV nucleic acid [ka]

[0165] In some embodiments, the antigen recognition domain of a CAR described herein comprises the complementarity determining regions (CDRs) and / or the heavy chain variable domain (VH) and the light chain variable domain (VL) from the anti-CD22 antibody m971. The m971 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 82 and a VL comprising the amino acid sequence of SEQ ID NO: 83. The amino acid sequences of the VH (and corresponding CDRH1, CDRH2, and CDRH3) and VL (and corresponding CDRL1, CDRL2, and CDRL3) of m971 are shown below. M971-VH: [ka] M971-VL: [ka] M971-CDRH1: GDSVSSNSAA (SEQ ID NO: 84) M971-CDRH2: TYYRSKWYN (SEQ ID NO: 85) M971-CDRH3:AREVTGDLEDAFDI (SEQ ID NO: 86) M971-CDRL1: QTIWSY (SEQ ID NO: 87) M971-CDRL2: AAS (SEQ ID NO: 88) M971-CDRL3: QQSYSIPQT (SEQ ID NO: 89)

[0166] In some embodiments, the antigen recognition domain of a CAR described herein comprises an scFv comprising a VH and a VL, where the VH comprises a CDRH1 of SEQ ID NO: 84, a CDRH2 of SEQ ID NO: 85, and a CDRH3 of SEQ ID NO: 86, and the VL comprises a CDRL1 of SEQ ID NO: 87, a CDRL2 of SEQ ID NO: 88, and a CDRL3 of SEQ ID NO: 89. In some embodiments, the antigen recognition domain of a CAR described herein comprises a VH and a VL, where the VH comprises the amino acid sequence of SEQ ID NO: 82, and the VL comprises the amino acid sequence of SEQ ID NO: 83.

[0167] The antigen recognition domain of the CAR provided herein may comprise CDRs and / or VH and VL derived from an anti-CD22 antibody (or antigen-binding fragment thereof). The anti-CD22 antibodies of the present disclosure can comprise any one of the partial light chain sequences known in the art and / or any one of the partial heavy chain sequences known in the art. In some embodiments, the antigen recognition domain of the CAR described herein comprises an scFv comprising a VH and a VL, where the VH comprises the amino acid sequence of a VH from an anti-CD22 antibody known in the art, and the VL comprises the amino acid sequence of the corresponding VL known in the art.

[0168] In some embodiments, the antigen recognition domain of the CAR described herein comprises an scFv comprising a VH and a VL, where the VH comprises CDRH1, CDRH2, and CDRH3, which comprise the amino acid sequences of CDRH1, CDRH2, and CDRH3, respectively, of an anti-CD22 antibody known in the art, and the VL comprises CDRL1, CDRL2, and CDRL3, which comprise the amino acid sequences of CDRL1, CDRL2, and CDRL3, respectively, of the same anti-CD22 antibody known in the art. Determination of CDR regions is well within the skill of one in the art. It is understood that in some embodiments, the CDRs can be a combination of Kabat CDRs and Chothia CDRs (also referred to as "combined CRs" or "extended CDRs").

[0169] In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other embodiments, the CDRs are IMGT CDRs. In other words, in embodiments having two or more CDRs, the CDRs may be any combination of Kabat, Chothia, IMGT CDRs, or combinations thereof.

[0170] ii) An antigen recognition domain comprising an anti-CD19 antibody or a fragment thereof In some embodiments, the antigen recognition domain of the CARs provided herein comprises an scFv that is affinity optimized for CD19 to induce cytotoxicity of tumor cells that produce high or normal levels of CD19. In some embodiments, the antigen recognition domain of the CARs provided herein comprises an scFv that is affinity optimized for CD19 to induce cytotoxicity of tumor cells that produce low levels of CD19. Examples of such affinity tuning are provided in Caruso et al. (2015) Cancer Res. 75:3505-18 and Liu et al. (2015) Cancer Res. 75:3596-607.

[0171] In some embodiments, the antigen recognition domain of the CARs provided herein comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen recognition domain of the CARs provided herein comprises a single-chain antibody fragment (scFv) comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) of a monoclonal anti-CD19 antibody. Optionally, the VH and VL may be linked by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In some embodiments, the scFv is humanized. In some embodiments, the antigen-binding portion may comprise a VH and VL directionally linked, for example, from N-terminus to C-terminus (VH-linker-VL or VL-linker-VH).

[0172] In some embodiments, the antigen recognition domain of the CARs provided herein comprises an scFv that is affinity optimized for CD19 to induce cytotoxicity of tumor cells that produce high or normal levels of CD19. In some embodiments, the antigen recognition domain of the CARs provided herein comprises an scFv that is affinity optimized for CD19 to induce cytotoxicity of tumor cells that produce low levels of CD19.

[0173] In some embodiments, the antigen recognition domain of a CAR provided herein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence of SEQ ID NO:90.

[0174] In some embodiments, the antigen recognition domain of a CAR provided herein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence of any one of SEQ ID NO:91.

[0175] Exemplary anti-CD19 scFvs from which the antigen recognition domain for use in the CARs described herein may be derived include, but are not limited to, FMC63 and its immunologically active and / or antigen-binding fragments. Thus, in some embodiments, the antigen recognition domain of the CARs provided herein comprises a VH and a VL derived from any one of the anti-CD19 antibodies FMC63.

[0176] Exemplary anti-CD19 scFvs from which the antigen recognition domain for use in the CARs described herein may be derived include, but are not limited to, inebilizumab (MEDI-551), MDX-1342, tafasitamab, obexelimab, B4 (Merck), hA19 (immunomedics), and immunologically active and / or antigen-binding fragments thereof. Thus, in some embodiments, the antigen recognition domain of the CARs provided herein comprises a VH and a VL derived from any one of these anti-CD19 antibodies.

[0177] In some embodiments, the antigen recognition domain of a CAR described herein comprises the complementarity determining regions (CDRs) and / or the heavy chain variable domain (VH) and the light chain variable domain (VL) from the anti-CD19 antibody FMC63. The FMC63 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 92 and a VL comprising the amino acid sequence of SEQ ID NO: 93. The amino acid sequences of the VH (and corresponding CDRH1, CDRH2, and CDRH3) and VL (and corresponding CDRL1, CDRL2, and CDRL3) of FMC63 are shown below. FMC63-VH: [ka] FMC63-VL: [ka] FMC63-CDRH1: GVSLPDYG (SEQ ID NO: 94) FMC63-CDRH2: IWGSETT (SEQ ID NO: 95) FMC63-CDRH3: AKHYYYGGSYAMDY (SEQ ID NO: 96) FMC63-CDRL1: QDISKY (SEQ ID NO: 97) FMC63-CDRL2: HTS (SEQ ID NO: 98) FMC63-CDRL3: QQGNTLPYT (SEQ ID NO: 99)

[0178] In some embodiments, the antigen recognition domain of a CAR described herein comprises an scFv comprising a VH and a VL, where the VH comprises a CDRH1 of SEQ ID NO: 94, a CDRH2 of SEQ ID NO: 95, and a CDRH3 of SEQ ID NO: 96, and the VL comprises a CDRL1 of SEQ ID NO: 97, a CDRL2 of SEQ ID NO: 98, and a CDRL3 of SEQ ID NO: 99. In some embodiments, the antigen recognition domain of a CAR described herein comprises a VH and a VL, where the VH comprises the amino acid sequence of SEQ ID NO: 92, and the VL comprises the amino acid sequence of SEQ ID NO: 93.

[0179] The antigen recognition domain of the CAR provided herein may comprise CDRs and / or VH and VL derived from an anti-CD19 antibody (or antigen-binding fragment thereof). The anti-CD19 antibodies of the present disclosure can comprise any one of the partial light chain sequences known in the art and / or any one of the partial heavy chain sequences known in the art. In some embodiments, the antigen recognition domain of the CAR described herein comprises an scFv comprising a VH and a VL, where the VH comprises the amino acid sequence of a VH derived from an anti-CD19 antibody known in the art, and the VL comprises the amino acid sequence of a corresponding VL derived from an anti-CD19 antibody known in the art.

[0180] In some embodiments, the antigen recognition domain of the CAR described herein comprises an scFv comprising a VH and a VL, where the VH comprises CDRH1, CDRH2, and CDRH3, which comprise the amino acid sequences of CDRH1, CDRH2, and CDRH3, respectively, of an anti-CD19 antibody known in the art, and the VL comprises CDRL1, CDRL2, and CDRL3, which comprise the amino acid sequences of CDRL1, CDRL2, and CDRL3, respectively, of the same anti-CD19 antibody known in the art. Determination of CDR regions is well within the skill of one in the art. It is understood that in some embodiments, the CDRs can be a combination of Kabat CDRs and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs").

[0181] In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDRs are Chothia CDRs. In other embodiments, the CDRs are IMGT CDRs. In other words, in embodiments having two or more CDRs, the CDRs may be any combination of Kabat, Chothia, IMGT CDRs, or combinations thereof.

[0182] B. Signal Peptides

[0183] In some embodiments, any of the CARs provided herein comprises a signal peptide (also known as a signal peptide, signal sequence, signal peptide sequence, leader peptide, and leader peptide sequence). In some embodiments, the antigen recognition domain of a CAR described herein comprises a signal peptide or a leader peptide sequence. Exemplary signal sequences include, but are not limited to, the CD8α signal sequence or an IgG signal sequence. In some embodiments, a CAR described herein does not comprise a signal peptide. In some embodiments, a T cell or population of T cells provided herein comprises a CAR that comprises a signal peptide. In some embodiments, a T cell or population of T cells provided herein comprises a CAR that does not comprise a signal peptide.

[0184] In some embodiments, the CAR (e.g., the antigen recognition domain of the CAR) comprises a human CD8α signal sequence comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1.

[0185] In some embodiments, the CAR (e.g., the antigen recognition domain of the CAR) comprises a human CD8α signal sequence comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:2.

[0186] In some embodiments, the CAR (e.g., the antigen recognition domain of the CAR) comprises a human IgG signal sequence comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:3.

[0187] In some embodiments, the CAR (e.g., the antigen recognition domain of the CAR) comprises a human IgG signal sequence comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:4.

[0188] C. Hinge domain In some embodiments, a hinge domain (also known as a spacer region or stalk region) is located between the antigen recognition domain and the transmembrane domain of the CAR. In particular, the stalk region is used to provide greater flexibility and accessibility to the extracellular antigen recognition domain. In some embodiments, the hinge domain may comprise up to about 300 amino acids. In some embodiments, the hinge comprises about 10 to about 100 amino acids in length. In some embodiments, the hinge comprises about 25 to about 50 amino acids in length. In some embodiments, the hinge domain establishes an optimal effector-target inter membrane distance. In some embodiments, the hinge domain provides flexibility for the antigen recognition domain to bind to the target antigen. Any protein that is stable and / or dimerizes can serve this purpose.

[0189] The hinge domain may be derived from all or part of a naturally occurring molecule, such as CD8, CD8α, CD4, CD28, 4-1BB, or all or part of the extracellular region of an IgG (particularly the hinge domain of an IgG, such as IgG1, IgG2, or IgG4), or all or part of a heavy chain constant region of an antibody. Alternatively, the hinge domain may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence. In some embodiments, it corresponds to an Fc domain of a human immunoglobulin, such as either the CH2 domain or the CH3 domain. In some embodiments, the CH2 and CH3 hinge domains of the human immunoglobulin are modified to improve dimerization. In some embodiments, the hinge is an immunoglobulin hinge portion. In some embodiments, the hinge domain comprises a CH3 region of a human immunoglobulin. In some implementations, the hinge domain comprises a CH2 and CH3 region of a human immunoglobulin. In some embodiments, the CH2 region comprises a human IgG1, IgG2, or IgG4 immunoglobulin CH2 region.

[0190] In some embodiments, the hinge domain is a portion of the human CD8α chain (e.g., NP_001139345.1). In some embodiments, the hinge domain of a CAR described herein comprises a subsequence of the constant region of CD8α, CD28, or an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4), either in wild-type form or mutated to avoid Fc receptor binding specifically to the hinge domain of either CD8α or CD28. In some embodiments, the stalk region comprises a human CD8α hinge or a human CD28 hinge.

[0191] In some embodiments, the hinge may comprise or consist of a human CD8α hinge domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:5.

[0192] In some embodiments, the hinge may comprise or consist of a human CD8α hinge domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:6.

[0193] In some embodiments, the hinge may comprise or consist of a human CD28 hinge domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:7.

[0194] In some embodiments, the hinge may comprise or consist of a human CD28 hinge domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:8.

[0195] D. Transmembrane Domain Suitable transmembrane domains of the CARs disclosed herein have the ability (a) to be expressed on the surface of a cell, which in some embodiments is an immune cell, such as a T cell, and / or (b) to interact with a ligand binding domain and an intracellular signaling domain to direct the cellular response of the immune cell to a given target cell. The transmembrane domain can be derived from either natural or synthetic sources. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the transmembrane domain can include the alpha, beta, delta, or gamma transmembrane region of the T cell receptor, or a transmembrane region from CD8, CD8α, CD8beta, CD28, CD3-epsilon, CD3-delta, CD3-gamma, CD3z, CD4, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, or BTLA transmembrane domain, or a portion of any of the foregoing, or a combination of any of the foregoing. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0196] Alternatively, the transmembrane domain can be synthetic and contain hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at one or both ends of the synthetic transmembrane domain. Optionally, a short oligonucleotide or polypeptide linker, in some embodiments 2-10 amino acids in length, may form the link between the transmembrane domain and the intracellular domain of the CAR. In some embodiments, the linker is a glycine-serine linker.

[0197] In some embodiments, the transmembrane domain of a CAR provided herein may comprise or consist of a human CD8α transmembrane domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 13.

[0198] In some embodiments, the transmembrane domain of a CAR provided herein may comprise or consist of a human CD28 transmembrane domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:14.

[0199] E. Costimulatory Domain The intracellular domain of the CAR provided herein may comprise one or more costimulatory domains. Exemplary costimulatory domains include, but are not limited to, 4-1BB (CD137), CD28, CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CD8α, OX40 (CD134), ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4 costimulatory domain, or fragments thereof, or combinations thereof. In some examples, the first CAR described herein comprises one or more or two or more costimulatory domains selected from 4-1BB (CD137), CD28, CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CD8α, OX40 (CD134), ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4 costimulatory domain, or fragments thereof, or combinations thereof. In some embodiments, the CAR described herein comprises a CD28 costimulatory domain or a fragment thereof. In some embodiments, the CAR described herein comprises a 4-1BB (CD137) costimulatory domain or a fragment thereof.

[0200] In some embodiments, the costimulatory domain of the CAR provided herein may comprise or consist of a human CD28 costimulatory domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:15.

[0201] In some embodiments, the costimulatory domain of the CAR provided herein may comprise or consist of a human CD28 costimulatory domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 16.

[0202] In some embodiments, the costimulatory domain of a CAR provided herein may comprise or consist of a human 4-1BB costimulatory domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:17.

[0203] F. Activation Domain In some embodiments, the activation domain of a CAR disclosed herein is responsible for activating at least one of the normal effector functions of an immune cell (e.g., immune cell) in which the CAR is expressed. The terms "intracellular signaling domain" or "intracellular domain" are used interchangeably and refer to a domain that includes a costimulatory domain and / or an activation domain. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, a helper activity, including cytolytic activity or secretion of cytokines. The term "activation domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Typically, the entire activation domain can be used, but often it is not necessary to use the entire chain. To the extent that a truncated portion of an activation domain is used, such a truncated portion may be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term activation domain is meant to include any truncated portion of the activation domain sufficient to transmit the effector function signal. In some embodiments, the activation domain further comprises a signaling domain for T cell activation. In some embodiments, the signaling domain for T cell activation comprises an intracellular domain derived from CD3ζ (CD3 zeta; CD3z) or an intracellular domain derived from LAT. In some embodiments, the CAR described herein comprises at least one (e.g., one, two, three, or more) activation domain selected from a CD3ε, or a LAT activation domain, or a portion of any of the foregoing. In some embodiments, the CAR described herein has an activation domain comprising a domain derived from CD3ζ (CD3 zeta; CD3z). In some embodiments, the CAR described herein has an activation domain comprising a domain derived from LAT.

[0204] In some embodiments, the activation domain of a CAR described herein may comprise or consist of a CD3 zeta activation domain (e.g., a human CD3 zeta activation domain) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:24.

[0205] In some embodiments, the activation domain of a CAR described herein may comprise or consist of a CD3 zeta activation domain (e.g., a human CD3 zeta activation domain) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:25.

[0206] In some embodiments, the CD3 zeta activation domain comprises a mutation in the ITAM domain. Examples of mutations in the ITAM domain of CD3 zeta are provided in Feucht et al., Nat Med. 2019;25(1):82-88. In some embodiments, each of two tyrosine residues in one or more of the ITAM1, ITAM2, or ITAM3 domains of the CD3 zeta activation domain is point mutated to a phenylalanine residue. In some embodiments, the CD3 zeta activation domain comprises one or more deletions of the ITAM1, ITAM2, or ITAM3 domains.

[0207] In some embodiments, the activation domain of a CAR described herein may comprise or consist of a LAT activation domain (e.g., a human LAT activation domain) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 26-34.

[0208] In some embodiments, the LAT activation domain comprises a mutation in a ubiquitination site.

[0209] In some embodiments, the activation domain of a CAR provided herein may comprise or consist of a LAT intracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:27.

[0210] In some embodiments, the activation domain of a CAR provided herein may comprise or consist of a LAT intracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:28.

[0211] In some embodiments, the activation domain of a CAR provided herein may comprise or consist of a LAT intracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:29.

[0212] In some embodiments, the activation domain of a CAR provided herein may comprise or consist of a LAT intracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:30.

[0213] In some embodiments, the activation domain of a CAR provided herein may comprise or consist of a LAT intracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:31.

[0214] In some embodiments, the activation domain of a CAR provided herein may comprise or consist of a LAT intracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 26, having a lysine to arginine substitution at position 25 of SEQ ID NO: 26 (K25R), a glycine to glutamic acid substitution at position 133 of SEQ ID NO: 26 (G133E), a lysine to arginine substitution at position 206 of SEQ ID NO: 26 (K206R), or any combination of the foregoing substitutions.

[0215] In some embodiments, the activation domain of a CAR provided herein may comprise or consist of a LAT intracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 32, having a lysine to arginine substitution at position 25 of SEQ ID NO: 32 (K25R), a glycine to glutamic acid substitution at position 104 of SEQ ID NO: 32 (G104E), a lysine to arginine substitution at position 177 of SEQ ID NO: 32 (K177R), or any combination of the foregoing substitutions.

[0216] In some embodiments, the activation domain of a CAR provided herein may comprise or consist of a LAT intracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 33, having a lysine to arginine substitution at position 25 of SEQ ID NO: 33 (K25R), a glycine to glutamic acid substitution at position 103 of SEQ ID NO: 33 (G103E), a lysine to arginine substitution at position 176 of SEQ ID NO: 33 (K176R), or any combination of the foregoing substitutions.

[0217] In some embodiments, the activation domain of a CAR provided herein may comprise or consist of a LAT intracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 34, having a lysine to arginine substitution at position 25 of SEQ ID NO: 34 (K25R), a glycine to glutamic acid substitution at position 132 of SEQ ID NO: 34 (G132E), a lysine to arginine substitution at position 205 of SEQ ID NO: 34 (K205R), or any combination of the foregoing substitutions.

[0218] The scope of the invention includes nucleic acid sequences encoding functional portions of the CARs described herein, including, for example, portions of a CAR that retain the ability to recognize target cells or detect, treat, or prevent disease to a similar, equal, or greater extent than the parent CAR.

[0219] In embodiments, the CARs described herein comprise additional amino acids at the amino or carboxy terminus or both termini of the moiety, which additional amino acids are not found in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional moiety, such as recognizing a target cell, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance the biological activity of the CAR compared to the biological activity of the parent CAR.

[0220] The term "functional variant" as used herein with respect to a CAR refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a CAR encoded by a nucleic acid sequence, where the functional variant retains the biological activity of the variant CAR. Functional variants include, for example, variants of a CAR described herein (parent CAR) that retain the ability to recognize a target cell to a similar extent, to the same extent, or to a greater extent than the parent CAR. With respect to the nucleic acid sequence encoding the parent CAR, the nucleic acid sequence encoding the functional variant of a CAR can be, for example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical to the nucleic acid sequence encoding the parent CAR.

[0221] The CARs described herein (including functional portions and functional variants thereof) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted to acid addition salts, and / or optionally dimerized or multimerized.

[0222] Table 8 provides exemplary amino acid sequences of domains that can be used in the CARs described herein. In some embodiments, the CARs provided herein comprise one or more domains described in Table 8, or fragments or portions thereof.

[0223] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0224] Table 9 provides exemplary nucleic acid sequences of domains that can be used to encode a CAR described herein. In some embodiments, a nucleic acid sequence encoding a CAR provided herein comprises one or more sequences described in Table 9, or a fragment or portion thereof.

[0225] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15]

[0226] G. Exemplary CAR Constructs i) Anti-CD22 CAR construct Disclosed herein is a CAR that specifically binds to CD22. In some embodiments, the CAR comprises an antigen recognition domain that specifically binds to human CD22, a hinge domain that comprises or consists of a CD8α hinge domain, a transmembrane domain that comprises or consists of a CD8α transmembrane domain, a costimulatory domain that comprises or consists of a 4-1BB costimulatory domain, and an intracellular signaling domain that comprises or consists of a CD3 zeta activation domain. Also disclosed herein is a nucleic acid sequence that encodes the CAR. In some embodiments, the T cells or population of T cells described herein are genetically modified to express at least one of the exemplary anti-CD22 CAR constructs described herein.

[0227] The amino acid sequence of an exemplary anti-CD22 CAR ("CAR1", "CD22 CAR", "second generation CAR", "second generation CD22 CAR", "2G CD22 CAR", "CD22 CART", "CD22BBz CAR", "CD22BBz", "second generation CD22BBz", "CD222-second generation CAR", "22BBz", "22SA", "22SAff", or "2G CAR") is shown below. (CD8α signal peptide, CD22 scFv(m971), CD8α hinge, CD8α transmembrane domain, 4-1BB signaling domain, CD3z signaling domain) [ka]

[0228] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:69.

[0229] The amino acid sequence of an exemplary anti-CD22 CAR ("CAR1", "CD22 CAR", "second generation CAR", "second generation CD22 CAR", "2G CD22 CAR", "CD22 CART", "CD22BBz CAR", "CD22BBz", "second generation CD22BBz", "CD222-second generation CAR", "22BBz", "22SA", "22SAff", or "2G CAR") is shown below. (CD8α signal peptide, CD22 scFv(m971), CD8α hinge, CD8α transmembrane domain, 4-1BB signaling domain, CD3z signaling domain) [ka]

[0230] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% percent identity to the amino acid sequence of SEQ ID NO: 102.

[0231] An exemplary anti-CD22 CAR, "CAR1", "CD22 CAR", "second generation CAR", or "2G CAR" polynucleotide sequence is shown below. (CD8α signal peptide, CD22 scFv(m971), CD8α hinge, CD8α transmembrane domain, 4-1BB signaling domain, CD3z signaling domain) [ka]

[0232] In some embodiments, the anti-CD22 CAR provided herein is encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO:70.

[0233] An exemplary anti-CD22 CAR, "CAR1", "CD22 CAR", "second generation CAR", or "2G CAR" polynucleotide sequence is shown below. (CD8α signal peptide, CD22 scFv(m971), CD8α hinge, CD8α transmembrane domain, 4-1BB signaling domain, CD3z signaling domain) [ka]

[0234] In some embodiments, the anti-CD22 CAR provided herein is encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 103.

[0235] Exemplary bicistronic anti-CD22 CARs and anti-CD22 CARs "CAR1-linker-CAR2" or "LAT-CAR" or "22ALA-CART" with wild-type LAT domain amino acid sequences are shown below (CAR1; furin / P2A linker; P2A linker; CAR2): [ka]

[0236] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 217.

[0237] Exemplary bicistronic anti-CD22 CARs and anti-CD22 CARs "CAR1-linker-CAR2" or "LAT-CAR" or "22ALA-CART" with a K52R mutation in the LAT domain amino acid sequence are shown below (CAR1; furin / P2A linker; CAR2, K52R): [ka]

[0238] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 218.

[0239] Exemplary bicistronic anti-CD22 CARs and anti-CD22 CARs "CAR1-linker-CAR2" or "LAT-CAR" or "22ALA-CART" with a K233R mutation in the LAT domain amino acid sequence are shown below (CAR1; furin / P2A linker; CAR2, K233R): [ka]

[0240] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 219.

[0241] Exemplary bicistronic anti-CD22 CARs and anti-CD22 CARs "CAR1-linker-CAR2" or "LAT-CAR" or "22ALA-CART" with K52R+K233R mutations in the LAT domain amino acid sequence are shown below (CAR1; furin / P2A linker; CAR2, K52R, K233R): [ka]

[0242] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 220.

[0243] Exemplary bicistronic anti-CD22 CARs and anti-CD22 CARs "CAR1-linker-CAR2" or "LAT-CAR" or "22ALA-CART" with K52R+G160E mutations in the LAT domain amino acid sequence are shown below (CAR1; furin / P2A linker; CAR2, K52R, G160E): [ka]

[0244] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 221.

[0245] Exemplary bicistronic anti-CD22 CARs and anti-CD22 CARs "CAR1-linker-CAR2" or "LAT-CAR" or "22ALA-CART" with K52R+K233R+G160E mutations in the LAT domain amino acid sequence are shown below (CAR1; furin / P2A linker; CAR2, K52R, K233R, G160E): [ka]

[0246] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 222.

[0247] ii) Exemplary Anti-CD19 CAR Constructs Disclosed herein is a CAR that specifically binds to CD19. In some embodiments, the CAR comprises an antigen recognition domain that specifically binds to human CD19, a hinge domain that comprises or consists of a CD28 hinge domain, a transmembrane domain that comprises or consists of a CD28 transmembrane domain, and an intracellular signaling domain that comprises or consists of a LAT intracellular signaling domain. Also disclosed herein is a nucleic acid sequence that encodes the CAR. In some embodiments, the T cells or population of T cells described herein are genetically modified to express at least one of the exemplary anti-CD19 CAR constructs described herein.

[0248] Exemplary bicistronic anti-CD19 CAR and anti-CD19 CAR "CAR1-linker-CAR2" or "LAT-CAR" or "19ALA-CART" amino acid sequences are shown below (CAR1; furin / P2A linker; CAR2): [ka]

[0249] In some embodiments, the anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 223.

[0250] Exemplary bicistronic anti-CD19 CAR and anti-CD19 CAR "CAR1-linker-CAR2" or "LAT-CAR" or "19ALA-CART" polynucleotide sequences are shown below. (CAR1; Furin / P2A linker; CAR2) [ka] [ka]

[0251] In some embodiments, the anti-CD19 CAR provided herein is encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 224.

[0252] The amino acid sequence of an exemplary anti-CD19 CAR, "CAR1", "CD19BBz", "second generation CD19 CAR", or "2G CD19 CAR", is shown below. (CD8α signal peptide, CD19 scFv(FMC63), CD8α hinge, CD8α transmembrane domain, 4-1BB signaling domain, CD3z signaling domain) [ka]

[0253] In some embodiments, the anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 225.

[0254] An exemplary anti-CD19 CAR "CAR2" or "CD19 CAR" amino acid sequence is shown below. (IgG signal peptide, CD19 scFv (FMC63), CD28 hinge, CD28 transmembrane domain, LAT signaling domain (mutated at K52R) [ka]

[0255] In some embodiments, the anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:71.

[0256] An exemplary anti-CD19 CAR "CAR2" or "CD19 CAR" amino acid sequence is shown below. (IgG signal peptide, CD19 scFv (FMC63), CD28 hinge, CD28 transmembrane domain, LAT signaling domain (with K52R mutation) [ka]

[0257] In some embodiments, the anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 100.

[0258] An exemplary anti-CD19 CAR "CAR2" or "CD19 CAR" polynucleotide sequence is shown below. (IgG signal peptide, CD19 scFv (FMC63), CD28 hinge, CD28 transmembrane domain, LAT signaling domain (mutated at K52R) [ka]

[0259] In some embodiments, the anti-CD19 CAR provided herein is encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO:72.

[0260] An exemplary anti-CD19 CAR "CAR2" or "CD19 CAR" polynucleotide sequence is shown below. (IgG signal peptide, CD19 scFv (FMC63), CD28 hinge, CD28 transmembrane domain, LAT signaling domain (mutated at K52R) [ka]

[0261] In some embodiments, the anti-CD19 CAR provided herein is encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 101.

[0262] A. Other Exemplary First CARs of the Present Disclosure

[0263] Exemplary Anti-CD19

[0264] [ka]

[0265] In some embodiments, the anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 309.

[0266] B. Other Exemplary Second CARs

[0267] Exemplary anti-CD22-LAT CAR

[0268] [ka]

[0269] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 300.

[0270] Exemplary anti-CD22-LAT-K52R CAR

[0271] [ka]

[0272] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 301.

[0273] Exemplary anti-CD22-LAT-K233R CAR

[0274] [ka]

[0275] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 302.

[0276] Exemplary anti-CD22-LAT-K52R-K233R CAR

[0277] [ka]

[0278] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 303.

[0279] Exemplary anti-CD22-LAT-K52R-G160E CAR

[0280] [ka]

[0281] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 304.

[0282] Exemplary anti-CD22-LAT-K52R-K233R-G160E CAR

[0283] [ka]

[0284] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 305.

[0285] Exemplary anti-CD22-HiAff-LAT CAR

[0286] [ka]

[0287] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 306.

[0288] Exemplary anti-CD19-LAT CAR

[0289] [ka]

[0290] In some embodiments, the anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 307.

[0291] Exemplary anti-CD22-SAff-LAT CAR

[0292] [ka]

[0293] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 308.

[0294] Cleavage sequence

[0295] The cleavage sequence can be used to generate linked or co-expression of genes in the constructs provided in the present disclosure. For example, the cleavage sequence can be used to co-express genes (e.g., CAR1 and CAR2) by linking open reading frames to form a single cistron (e.g., bicistronic CAR). In some aspects, the cleavage sequence can include a 2A self-cleaving peptide sequence element. Exemplary 2A self-cleaving peptide sequence elements include, but are not limited to, T2A, P2A, E2A, and F2A. In some embodiments, the cleavage sequence includes a P2A sequence. In some embodiments, the cleavage sequence can include a furin cleavage peptide. In some embodiments, the cleavage sequence can include a furin cleavage peptide and a P2A sequence.

[0296] In some embodiments, P2A comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:73).

[0297] In some embodiments, P2A comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:74).

[0298] In some embodiments, T2A comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of GSGEGRGSLTCGDVEENPGP (SEQ ID NO: 75).

[0299] In some embodiments, E2A comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:76).

[0300] In some embodiments, F2A comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:77).

[0301] In some embodiments, the furin cleavage peptide comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of RKRRGSGTPDPW (SEQ ID NO:78).

[0302] In some embodiments, the cleavage sequence comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of RKRRGSGTPDPWGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:79).

[0303] In some embodiments, the CAR described herein can be under the control of an inducible promoter of gene transcription.In some embodiments, the inducible promoter is an EF1a promoter.In some embodiments, the inducible promoter is a PGK promoter.

[0304] iii) Exemplary Bicistronic CAR Constructs Exemplary sequences of constructs disclosed herein, including anti-CD22 CARs and anti-CD19 CARs, are shown below.

[0305] Exemplary bicistronic anti-CD22 CAR and anti-CD19 CAR "CAR1-linker-CAR2" or "LAT-CAR" amino acid sequences are shown below (CAR1; furin / P2A linker; CAR2). [ka]

[0306] In some embodiments, the bicistronic anti-CD22 CAR and anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:80.

[0307] Exemplary bicistronic anti-CD22 CAR and anti-CD19 CAR "CAR1-linker-CAR2" or "LAT-CAR" or "ALA-CART" or "22x19ALA-CART" or "ALA-CART CD22BBz" or "CD22 second generation CAR+CD19-LAT CAR" or "22x19LAT" amino acid sequences are shown below (CAR1; furin / P2A linker; CAR2). [ka]

[0308] In some embodiments, the bicistronic anti-CD22 CAR and anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 104.

[0309] Exemplary bicistronic anti-CD22 CAR and anti-CD19 CAR "CAR1-linker-CAR2" or "LAT-CAR" polynucleotide sequences are shown below. (CAR1; Furin / P2A linker; CAR2) [ka] [ka]

[0310] In some embodiments, the bicistronic anti-CD22 CAR and anti-CD19 CAR provided herein are encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO:81.

[0311] In some embodiments, the bicistronic anti-CD22 CAR and anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 104.

[0312] Exemplary bicistronic anti-CD22 CAR and anti-CD19 CAR "CAR1-linker-CAR2" or "LAT-CAR" polynucleotide sequences are shown below. (CAR1; Furin / P2A linker; CAR2) [ka] [ka]

[0313] In some embodiments, the bicistronic anti-CD22 CAR and anti-CD19 CAR provided herein are encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 105.

[0314] iii) Exemplary anti-CD22 bicistronic affinity CAR constructs

[0315] The amino acid sequences of exemplary bicistronic standard affinity anti-CD22 CARs and standard affinity anti-CD22 LAT-CAR "CAR1-linker-CAR2" or "SAff / SAff-LAT" or "LAT-CAR" or "22ALACART1" are shown below (SAff scFv CAR1; furin / P2A linker; SAff scFv CAR2).

[0316] [ka]

[0317] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 200.

[0318] Exemplary bicistronic standard affinity anti-CD22 CAR and standard affinity anti-CD22 LAT-CAR "CAR1-linker-CAR2" or "SAff / SAff-LAT" or "LAT-CAR" or "22ALACART1" polynucleotide sequences are shown below (SAff scFv CAR1; SAff scFv CAR2)

[0319] [ka] [ka]

[0320] In some embodiments, the anti-CD22 CAR provided herein is encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO:201.

[0321] Exemplary bicistronic standard affinity anti-CD22 CAR and high affinity anti-CD22 LAT-CAR "CAR1-linker-CAR2" or "SAff / HiAff-LAT" or "LAT-CAR" or "22ALACART2" amino acid sequences are shown below (SAff scFv CAR1; Furin / P2A linker; HiAff scFv CAR2).

[0322] [ka]

[0323] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 202.

[0324] Exemplary bicistronic standard affinity anti-CD22 CAR and high affinity anti-CD22 LAT-CAR "CAR1-linker-CAR2" or "SAff / HiAff-LAT" or "LAT-CAR" or "22ALACART2" polynucleotide sequences are shown below (SAff scFv CAR1; HiAff scFv CAR2).

[0325] [ka] [ka]

[0326] In some embodiments, the anti-CD22 CAR provided herein is encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 203.

[0327] Exemplary bicistronic high affinity anti-CD22 CAR and standard affinity anti-CD22 LAT-CAR "CAR1-linker-CAR2" or "HiAff / SAff-LAT" or "LAT-CAR" or "22ALACART3" amino acid sequences are shown below (HiAff scFv CAR1; Furin / P2A linker; SAff scFv CAR2):

[0328] [ka]

[0329] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 204.

[0330] Exemplary bicistronic high affinity anti-CD22 CAR and standard affinity anti-CD22 LAT-CAR "CAR1-linker-CAR2" or "HiAff / SAff-LAT" or "LAT-CAR" or "22ALACART3" polynucleotide sequences are shown below (HiAff scFv CAR1; SAff scFv CAR2)

[0331] [ka] [ka]

[0332] In some embodiments, the anti-CD22 CAR provided herein is encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 205.

[0333] The amino acid sequences of exemplary bicistronic high affinity anti-CD22 CARs and high affinity anti-CD22 LAT-CARs "CAR1-linker-CAR2" or "HiAff / HiAff-LAT" or "LAT-CAR" or "22ALACART4" or "22ALA-CART" or "22ALACART" are shown below (HiAff scFv CAR1; Furin / P2A linker; HiAff scFv CAR2):

[0334] [ka]

[0335] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 206.

[0336] Exemplary bicistronic high affinity anti-CD22 CAR and high affinity anti-CD22 LAT-CAR "CAR1-linker-CAR2" or "HiAff / HiAff-LAT" or "LAT-CAR" or "22ALACART4" or "22ALACART" polynucleotide sequences are shown below (HiAff scFv CAR1; HiAff scFv CAR2):

[0337] [ka] [ka]

[0338] In some embodiments, the anti-CD22 CAR provided herein is encoded by a polynucleotide sequence comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 207.

[0339] iv) Exemplary First CARs

[0340] Exemplary Anti-CARCD19 CARs

[0341] [ka]

[0342] In some embodiments, the anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 309.

[0343] v) Exemplary Second CA

[0344] Exemplary anti-CD22-LAT CAR

[0345] [ka]

[0346] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 300.

[0347] Exemplary anti-CD22-LAT-K52R CAR

[0348] [ka]

[0349] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 301.

[0350] Exemplary anti-CD22-LAT-K233R CAR

[0351] [ka]

[0352] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 302.

[0353] Exemplary anti-CD22-LAT-K52R-K233R CAR

[0354] [ka]

[0355] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 303.

[0356] Exemplary anti-CD22-LAT-K52R-G160E CAR

[0357] [ka]

[0358] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 304.

[0359] Exemplary anti-CD22-LAT-K52R-K233R-G160E CAR

[0360] [ka]

[0361] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 305.

[0362] Exemplary anti-CD22-HiAff-LAT CAR

[0363] [ka]

[0364] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 306.

[0365] Exemplary anti-CD19-LAT CAR

[0366] [ka]

[0367] In some embodiments, the anti-CD19 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 307.

[0368] Exemplary anti-CD22-SAff-LAT CAR

[0369] [ka]

[0370] In some embodiments, the anti-CD22 CAR provided herein may comprise or consist of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 308.

[0371]

[0372]

[0373] 4.CAR expression level

[0374] The present disclosure provides a population of engineered T cells, where a plurality of engineered T cells of the population comprises any of the chimeric stimulating receptors (CARs) disclosed herein. The present disclosure also provides a composition comprising a population of T cells, where a plurality of T cells of the population comprises a non-natural CAR that comprises, consists essentially of, or consists of a) a first chimeric antigen receptor (CAR) comprising an antigen recognition domain that binds a first antigen, a transmembrane domain, and an intracellular signaling domain, b) a second CAR comprising an antigen recognition domain that binds a second antigen, a transmembrane domain, and a linker for activation of T cells (LAT) intracellular signaling domain. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprises a first CAR and a second CAR. In some embodiments, each CAR polypeptide is expressed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 copies per cell. In some embodiments, the nucleic acid encoding the CAR is integrated into the genome at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 copies per cell.

[0375] In some embodiments, the ratio of CAR1:CAR2 copy numbers is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0376] 5. Antigen

[0377] In some embodiments, provided herein is a cell (e.g., a T cell) expressing a first CAR that targets a first antigen (e.g., anti-CD22) and a second CAR that targets a second antigen (e.g., anti-CD19).

[0378] Among the antigens that can be targeted by engineered antigen receptors are those expressed in association with the disease, condition, or cell type targeted by adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, malignant diseases and disorders, including cancers and tumors, including hematological cancers, cancers of the immune system, such as lymphomas, leukemias, and / or myelomas (e.g., B-cell, T-cell, and myeloid leukemias, lymphomas, and multiple myelomas). In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, such as tumor cells or pathogenic cells, compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0379] Any suitable antigen may be used in the present methods. Exemplary antigens include antigenic molecules from infectious pathogens, glycosylated antigens, TnAntigens, self / autoantigens, tumor / cancer In certain embodiments, the antigens include those listed in Table 1.

[0380] In certain embodiments, two or more Antigen Recognition Domain Antigens for targeting by include, but are not limited to, CD22 and CD19 (e.g., for B cell malignancies). The sequences of these antigens are Field of the Invention CD22 (e.g., accession number NM_001772.4); CD19 (e.g., accession number NC_000023.11).

[0381] Tumor-associated antigens may be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma, ovarian cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3, and MAGE4 (or other MAGE antigens such as those disclosed in PCT Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. 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), prostatic acid phosphatase, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

[0382] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, tumor antigens may be self-peptide hormones, such as full-length gonadotropin releasing hormone (GnRH), short peptides of 10 amino acids in length, which are useful in the treatment of many cancers.

[0383] Tumor antigens include tumor antigens from cancers characterized by tumor-associated antigen expression, such as expression of HER-2 / ne. Tumor-associated antigens of interest include lineage-specific tumor antigens, such as the melanocyte-melanoma lineage antigen MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-related proteins. Exemplary tumor-associated antigens include p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, , MC1R, gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CEA, CDK-4 / m, ELF2M, GnT-V, G2 50, 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 signal transducer 1 (TACSTD1) TACSTD2, receptor thymocyte activator (TACSTD2) tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (particularly EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2),Nuclear factor kappa B (NF-B), Notch receptors (e.g., Notchl-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and its regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, These include, but are not limited to, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and tumor antigens derived from or including any one or more of the idiotypes.

[0384] Antigens can 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 enzyme, survivin, mesothelin, mutated ras, bcr / abl rearrangements, Her2 / neu, mutated or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intronic sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens that include epitope regions or epitope peptides derived from tumor viral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; non-mutated oncofetal proteins with tumor selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0385] In other embodiments, antigens are obtained or derived from pathogenic or opportunistic pathogenic microorganisms (also referred to herein as infectious disease microorganisms), such as viruses, fungi, parasites, and bacteria, hi certain embodiments, antigens derived from such microorganisms include full-length proteins.

[0386] Exemplary pathogenic organisms for which antigens are contemplated for use in the methods described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B, and C, vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and JC virus), adenovirus, Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species, including Streptococcus pneumoniae. As will be appreciated by those of skill in the art, proteins from these and other pathogenic microorganisms for use as antigens as described herein, as well as nucleotide sequences encoding the proteins, can be identified in publications and public databases such as GENBANK®, SWISS-PROT®, and TREMBL®.

[0387] Antigens derived from Human Immunodeficiency Virus (HIV) include HIV virion structural proteins (e.g., gp120, gp41, pl7, p24), protease, reverse transcriptase, or any of the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0388] Antigens from herpes simplex viruses (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. The late gene group primarily encodes proteins that form virion particles. Such proteins include five proteins from (UL) that form the viral capsid: UL6, UL18, UL35, UL38, and major capsid proteins UL19, UL45, and UL27, each of which may be used as an antigen as described herein. Other exemplary HSV proteins contemplated for use as antigens herein include ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD) proteins. The HSV genome contains at least 74 genes that each encode a protein that may potentially be used as an antigen.

[0389] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed during the very early and early stages of viral replication, glycoproteins I and III, capsid proteins, coat proteins, lower matrix proteins pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products from the gene cluster from UL128-UL150 (Rykman et al. 2006), envelope glycoproteins B (gB), gH, gN, and ppl50. As will be appreciated by those of skill in the art, CMV proteins for use as antigens described herein can be identified in public databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see, e.g., Bennekov et al. 2004; Loewendorf et al. 2010; Marschall et al. 2009).

[0390] Antigens derived from Epstein-Barr virus (EBV) contemplated for use in certain embodiments include EBV lysis proteins gp350 and gp110, EBV proteins produced during latent cycle infection including Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), and latent membrane protein (LMP)-1, LMP-2A, and LMP-2B (see, e.g., Lockey et al., 2008).

[0391] Antigens from respiratory syncytial virus (RSV) contemplated for use herein include any of 11 proteins or antigenic fragments thereof encoded by the RSV genome: NS1, NS2, N (nucleocapsid protein), M (matrix protein) SH, G and F (viral coat proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcriptional control), RNA polymerase, and phosphoprotein P.

[0392] Antigens from vesicular stomatitis virus (VSV) contemplated for use include any one of the five major proteins and antigenic fragments thereof encoded by the VSV genome: the large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, e.g., Rieder et al., 1999).

[0393] Antigens derived from influenza virus contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[0394] Exemplary viral antigens also include adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigen), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (including hepatitis B core or surface antigen, hepatitis C virus E1 or E2 glycoproteins, core, or nonstructural proteins), herpesvirus polypeptides (including herpes simplex virus or varicella zoster virus glycoproteins), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, and the like. Examples of polypeptides that may be used include, but are not limited to, rabies virus polypeptides, orthomyxovirus polypeptides, papillomavirus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides (e.g., poliovirus capsid polypeptides), poxvirus polypeptides (e.g., vaccinia virus polypeptides), rabies virus polypeptides (e.g., rabies virus glycoprotein G), reovirus polypeptides, retrovirus polypeptides, and rotavirus polypeptides.

[0395] In certain embodiments, the antigen may be a bacterial antigen. In certain embodiments, the bacterial antigen of interest may be a secreted polypeptide. In other certain embodiments, the bacterial antigen includes an antigen having a portion of a polypeptide exposed on the extracellular surface of the bacteria.

[0396] Antigens derived from Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), that are contemplated for use include the Agr system, Sar and Sae, the Arl system, Sar homologs (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ, and TcaR), the Srr system, and virulence regulators such as TRAP. Other staphylococcal proteins that may serve as antigens include Clp proteins, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes of two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example, in PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As will be appreciated by those skilled in the art, staphylococcal proteins for use as antigens can also be identified in other public databases, such as GenBank®, Swiss-Prot®, and TrEMBL®.

[0397] Antigens from Streptococcus pneumoniae contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA, RrgB, RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as antigens in some embodiments (see, e.g., Zysk et al., 2000). The complete genome sequence of virulent strains of Streptococcus pneumoniae has been sequenced, and as will be appreciated by those of skill in the art, Streptococcus pneumoniae proteins for use herein may also be identified in other public databases, such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest for antigens according to the present disclosure include virulence factors and proteins predicted to be exposed on the surface of S. pneumoniae (see, e.g., Frolet et al., 2010).

[0398] Examples of bacterial antigens that can be used as antigens include Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae (H. influenzae type b outer membrane protein), Helicobacter polypeptides, Klebsiella polypeptides, L-type bacterial polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e., S. pneumoniae polypeptides) (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, Group A Streptococcus polypeptides (e.g., S. pyogenes M protein), Group B Streptococcus (S. agalactiae (S.agalactiae polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Y pestis F1 and V antigens).

[0399] Examples of fungal antigens include Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudomicrodochium polypeptides, Pythium polypeptides, Rhino Examples of polypeptides include, but are not limited to, sporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

[0400] 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, Plasmodium polypeptides. Helminth parasite antigens include Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius Polypeptides, Filaroides Polypeptides, Haemonchus Polypeptides, Lagochilascaris Polypeptides, Loa Polypeptides, Mansonella Polypeptides, Muellerius Polypeptides, Nanophyetus Polypeptides, Necator Polypeptides, Nematodirus Polypeptides, Oesophagostomum Polypeptides, Onchocerca Polypeptides, Opisthorchis Polypeptides, Ostertagia Polypeptides, Parafilaria Polypeptides, Paragonimus Polypeptides, Parascaris Polypeptides, Physaloptera Polypeptides, Protostrongylus Polypeptides,Examples of polypeptides that may be used include, but are not limited to, Setaria polypeptides, Spirocerca polypeptides, Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Tricho strongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides (e.g., P. falciparum circumsporozoite (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSAl c-term), and exported protein 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides.

[0401] Examples of ectoparasite antigens include, but are not limited to, fleas; mites, including hard and soft mites; flies, such as midges, mosquitoes, sandflies, black flies, horseflies, horn flies, deer flies, tsetse flies, stable flies, flies that cause myiasis, and biting gnats; ants; spiders, lice; mites; and polypeptides (including antigens and allergens) from hemipteran insects such as bedbugs and assassin bugs.

[0402] 6. Safety Switch Proteins Although cell therapy shows great promise for the treatment of human diseases, significant toxicity from the cells themselves or their transgene products has hindered clinical studies. In some embodiments described herein, immune effector cells (e.g., T cells) containing the CARs described herein that are infused into a mammalian subject, e.g., a human, can be removed to modulate the effect of such immune effector cells if toxicity results from their use. In some embodiments, the immune cells of the present disclosure may contain one or more suicide genes.

[0403] As used herein, the terms "safety switch protein," "suicide protein," or "kill switch protein" refer to engineered proteins designed to prevent potential toxicity or other adverse effects of cell therapy. In some cases, expression of the safety switch protein is conditionally controlled to address safety concerns of engineered transplanted cells that have a gene encoding the safety switch protein permanently integrated into their genome. This conditional regulation can vary and can include post-translational activation via small molecules and control by tissue-specific and / or transient transcriptional regulation. The safety switch can mediate induction of apoptosis, inhibition of protein synthesis or DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated exhaustion. In some cases, the safety switch protein is activated by an exogenous molecule, for example, a prodrug that, when activated, causes apoptosis and / or cell death of the therapeutic cell.

[0404] The term "suicide gene" or "kill switch gene" as used herein is defined as a gene that, upon administration of a prodrug, results in the transfer of the gene product to a compound that kills the host cell. Examples of suicide gene / prodrug combinations that can be used include, but are not limited to, inducible caspase 9 (iCASP9) and rimiduside; RQR8 and rituximab; truncated forms of EGFR variant III (EGFRv3) and cetuximab; herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase, thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. E. coli purine nucleoside phosphorylase, a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine. Other examples of suicide genes used in prodrug therapy are the E. coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0405] Exemplary suicide genes include, but are not limited to, inducible caspase 9 (or caspase 3 or 7), CD20, CD52, EGFRt, or thymidine kinase, cytosine deaminase, HER1, and any combination thereof. Additional suicide genes known in the art that may be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-alpha, Y-lyase (MET), and thymidine phosphorylase (TP).

[0406] 7.T cell activity In some embodiments, the population of engineered T cells disclosed herein exhibits a T cell function (e.g., effector function). In some embodiments, the population is cytotoxic to CD22-expressing cells and CD19-expressing cells (e.g., CD22 positive tumor cells, CD22 low tumor cells, CD19 positive tumor cells, CD19 low tumor cells). The effector function of the T cells can be, for example, a helper activity including cytolytic activity or secretion of cytokines. In some embodiments, the population exhibits one or more T cell effector functions at a level at least 3-4 fold higher than the function exhibited by a population of T cells that does not express a CAR.

[0407] III. Method

[0408] The chimeric antigen receptor can be easily inserted into and expressed by immune cells (e.g., T cells). In certain embodiments, the cells (e.g., immune cells such as T cells) are obtained from a donor subject. In some embodiments, the donor subject is a human patient suffering from cancer or a tumor. In other embodiments, the donor subject is a human patient not suffering from cancer or a tumor. In some embodiments, the engineered cells are autologous to the subject. In some embodiments, the engineered cells are allogeneic to the subject.

[0409] The cells of the present disclosure may be obtained through any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Additionally, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. In certain embodiments, cells collected from apheresis are washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. As will be appreciated, a washing step can be used, such as by using a semi-automated flow-through centrifuge, such as the Cobe™ 2991 cell processor, Baxter CytoMate™, and the like. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, undesirable components of the apheresis sample are removed. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0410] In certain embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, by using centrifugation through a PERCOLL™ gradient. + , CD8 + , CD28 + , CD45RA + , and CD45RO +Specific subpopulations of T cells, such as T cells, are further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. In some embodiments, cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells can be used. For example, negative selection can be used to enrich for CD4 + To enrich for cells, monoclonal antibody cocktails typically include antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In certain embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the present disclosure.

[0411] In some embodiments, PBMCs are used directly for genetic modification with immune cells (such as CAR or TCR) using the methods described herein. In certain embodiments, after isolating PBMCs, T lymphocytes are further isolated and both cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subpopulations before or after genetic modification and / or expansion.

[0412] In some embodiments, CD8 + The cells were then divided into naive, central memory, and effector cells, each of which expressed CD8 + Further classification is achieved by identifying cell surface antigens associated with the cells. In some embodiments, expression of phenotypic markers of central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and are negative for granzyme B. In some embodiments, central memory T cells are negative for CD8 + , CD45RO + , and CD62L +In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In certain embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. + T cells are further divided into subpopulations. For example, CD4 + T helper cells can be classified into naive cells, central memory cells, and effector cells by identifying cell populations that have cell surface antigens.

[0413] In some embodiments, immune cells, e.g., T cells, are genetically modified after isolation using known methods, or immune cells are activated and expanded (or differentiated in the case of progenitor cells) in vitro before being genetically modified. In another embodiment, immune cells, e.g., T cells, are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, and 6,797,514, and PCT Publication No. WO2012 / 079000, the contents of which are incorporated herein by reference in their entirety. In general, such methods include contacting PBMCs or isolated T cells with stimulatory and co-stimulatory agents, typically anti-CD3 and anti-CD28 antibodies bound to beads or other surfaces, in a medium containing appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies bound to the same bead function as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other embodiments, T cells are activated and stimulated to proliferate using feeder cells and appropriate antibodies and cytokines using methods described in U.S. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO2012 / 129514, which are incorporated by reference in their entireties.

[0414] IV. Methods of Gene Delivery and Cell Modification Those of skill in the art will be well-equipped to construct vectors by standard recombinant techniques for expression of the antigen receptors of the present disclosure (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference). Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors, including their replication-competent, replication-deficient, and These include, but are not limited to, vectors derived from human adenoviruses (including gutless forms), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, Maraba virus vectors, and group B adenovirus enadenotucirev vectors.

[0415] 1. Viral Vectors Viral vectors encoding antigen receptors, cytokines, and / or functional effector elements may be provided in certain embodiments of the disclosed methods. In generating recombinant viral vectors, non-essential genes are typically replaced with genes or coding sequences for heterologous (or non-native) proteins. Viral vectors are a type of expression construct that utilizes viral sequences to introduce nucleic acids and potentially proteins into cells. The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis and integrate into the host cell genome to stably and efficiently express viral genes makes them attractive candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that may be used to deliver nucleic acids of certain embodiments of the present invention are described below.

[0416] The engineered viral vector may include a long terminal repeat (LTR), a cargo nucleotide sequence, or a cargo cassette. As used herein, "cargo cassette" in relation to a viral vector refers to a nucleotide sequence that includes a left LTR at the 5' end and a right LTR at the 3' end, and a nucleotide sequence located between the left LTR and the right LTR. The nucleotide sequence flanking the LTR is the nucleotide sequence intended for integration into the acceptor DNA. "Cargo nucleotide sequence" refers to a nucleotide sequence flanked by LTRs at each end (e.g., a nucleotide sequence intended for integration into the acceptor DNA), where the LTR is heterologous to the nucleotide sequence. The cargo cassette can be engineered artificially.

[0417] In some embodiments of the method of the present disclosure, the introduction of the nucleic acid sequence and / or genome editing construct into the immune cell ex vivo, in vivo, in vitro, or in situ includes a viral vector. In some embodiments, the viral vector is a non-integrated non-chromosomal vector. Examples of non-integrated non-chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and herpes virus. In some embodiments, the viral vector is an integrated chromosomal vector. Integrated chromosomal vectors include, but are not limited to, adeno-associated vector (AAV), lentivirus, and gamma retrovirus.

[0418] Lentiviruses 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, e.g., U.S. Patent Nos. 6,013,516 and 5,994,136).

[0419] The retroviral vector may be, for example, a gamma retroviral vector. The gamma retroviral vector may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (for example, two) long terminal repeats (LTRs), and a transgene of interest, for example, a gene encoding a CAR. The gamma retroviral vector may lack viral structural genes such as gag, pol, env, etc. Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen focus forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., Viruses. 2011 Jun;3(6):677-713.

[0420] Recombinant lentiviral vectors can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses capable of infecting non-dividing cells, in which a suitable host cell is transfected with two or more vectors having packaging functions, i.e., gag, pol, and env, and rev and tat, are described in U.S. Patent No. 5,994,136 (herein incorporated by reference).

[0421] In some embodiments of the disclosed methods, the ex vivo, in vivo, in vitro, or in situ introduction of the nucleic acid sequence and / or genome editing construct into the immune cell includes a combination of vectors. Exemplary, non-limiting vector combinations include a viral vector and a non-viral vector, multiple non-viral vectors, or multiple viral vectors. Exemplary, but non-limiting vector combinations include a DNA-derived vector and an RNA-derived vector, a RNA-derived vector and a reverse transcriptase, a transposon and a transposase, a non-viral vector and an endonuclease, and a viral vector and an endonuclease.

[0422] In some embodiments of the disclosed methods, the genome modification comprises introducing a nucleic acid sequence and / or a genome editing construct into an immune cell ex vivo, in vivo, in vitro, or in situ, stably integrating the nucleic acid sequence, transiently integrating the nucleic acid sequence, resulting in site-specific integration of the nucleic acid sequence, or resulting in biased integration of the nucleic acid sequence. In some embodiments, the nucleic acid sequence is a transgene.

[0423] In some embodiments of the disclosed methods, the genome modification comprises introducing a nucleic acid sequence and / or a genome editing construct into an immune cell ex vivo, in vivo, in vitro, or in situ, stably integrating the nucleic acid sequence. In some embodiments, the stable chromosomal integration can be random integration, site-specific integration, or biased integration. In some embodiments, the site-specific integration can be non-assisted or assisted. In some embodiments, the site-specific integration assistance is co-delivered with a site-specific nuclease. In some embodiments, the site-specific nuclease comprises a transgene having 5' and 3' nucleotide sequence extensions that include percent homology to the upstream and downstream regions of the genome integration site. In some embodiments, the transgene having homologous nucleotide extensions allows genome integration by homologous recombination, microhomology-mediated end joining, or non-homologous end joining. In some embodiments, the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites can provide for the integration of new genetic material in a manner that ensures that the newly inserted genetic element is functional (e.g., expressed at therapeutically effective expression levels) and does not cause deleterious changes to the host genome that pose a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences in the human albumin gene, adeno-associated virus site 1 (AAVS1), the naturally occurring integration site of the AAV virus on chromosome 19, the chemokine (CC motif) receptor 5 (CCR5) gene, as well as sites in the human ortholog of the mouse Rosa26 locus.

[0424] In some embodiments, site-specific transgene integration occurs at a site that disrupts expression of a target gene. In some embodiments, disruption of target gene expression occurs by site-specific integration at an intron, exon, promoter, genetic element, enhancer, suppressor, start codon, stop codon, and response element. In some embodiments, exemplary target genes targeted by site-specific integration include, but are not limited to, any immunosuppressive gene and genes involved in allogeneic rejection.

[0425] In some embodiments, site-specific transgene integration occurs at a site that results in enhanced expression of the target gene, hi some embodiments, enhanced expression of the target gene occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0426] A. Regulatory Elements The expression cassette contained in the vector useful in the present disclosure specifically includes a eukaryotic transcriptional promoter operably linked (5' to 3' direction) to a protein coding sequence, a splice signal including intervening sequences, and a transcription termination / polyadenylation sequence. The promoters and enhancers that control the transcription of protein encoding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery can collect and integrate the regulatory information conveyed by each element, which allows different genes to evolve distinct and often complex transcriptional regulation patterns. Promoters used in the context of the present disclosure include constitutive promoters, inducible promoters, and tissue-specific promoters.

[0427] (i) Promoter / enhancer The expression constructs provided herein include a promoter that drives expression of the antigen receptor. Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the promoters of the SV40 late genes, separate elements that cover the start site itself serve to fix the start position. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although many promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control of a promoter," the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[0428] Spacing between promoter elements is often flexible, thus preserving promoter function when elements are inverted or moved relative to one another. In the tk promoter, spacing between promoter elements can be increased by up to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either in concert or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0429] A promoter may be one that is naturally associated with a nucleic acid sequence, as obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of the nucleic acid sequence. Alternatively, certain advantages are obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are "non-naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the lactamase (penicillinase), lactose and tryptophan (trp-) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, recombinant cloning and / or nucleic acid amplification techniques, including PCR™, may be used to produce the sequences in connection with the compositions disclosed herein. It is further contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be used as well.

[0430] Of course, it is important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, which may be advantageous, for example, in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0431] Additionally, any promoter / enhancer combination may be used to drive expression (e.g., according to the Eukaryotic Promoter Database EPDB on the World Wide Web at epd.isb-sib.ch / ). Use of the T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0432] Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoters, the cytomegalovirus (CMV) immediate early promoter, the Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, such as the beta-actin promoter, the GADPH promoter, the metallothionein promoter; and concatenated response element promoters, such as the cyclic AMP response element promoter (ere), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the response element promoter near the minimal TATA box (tre). It is also possible to use a human growth hormone promoter sequence (e.g., the human growth hormone minimal promoter described in Genbank, Accession No. X05244, nucleotides 283-341, or the mouse mammary tumor promoter (available from ATCC catalog number ATCC45007). In certain embodiments, the promoter is EF1, EF1α, MND, CMVIE, Dectin-1, Dectin-2, human CD1 lc, F4 / 80, SM22, RSV, SV40, AdMLP, β-actin, MHC class I, MHC class II promoter, U6 promoter, or H1 promoter, although any other promoter useful for driving expression of a therapeutic gene is applicable to the practice of the present disclosure.

[0433] In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e. nucleic acid sequences that increase the activity of a promoter and have the potential to act in cis, regardless of their orientation, even at relatively long distances (up to several kilobases away from the target promoter), however, the function of an enhancer is not necessarily limited to such long distances and can also function in the immediate vicinity of a particular promoter.

[0434] (ii) Initiation signal and ligated expression For efficient translation of coding sequences, specific initiation signals may also be used in the expression constructs provided in this disclosure. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translational control signals, including the ATG initiation codon. One of ordinary skill in the art would be able to easily determine this and provide the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons may be either natural or synthetic. Expression efficiency may be enhanced by including appropriate transcriptional function effector elements.

[0435] In certain embodiments, internal ribosome entry site (IRES) elements are used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of translation that relies on a 5' methylated cap and initiate translation at an internal site. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, along with IRESs from mammalian messages. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together to create polycistronic messages. IRES elements allow each open reading frame access to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0436] Additionally, certain 2A sequence elements can be used to effect linked or co-expression of genes in constructs provided herein. For example, a truncation sequence can be used to link open reading frames to form a single cistron, thereby allowing genes to be co-expressed. Exemplary truncation sequences are F2A (foot and mouth disease virus 2A) or "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A) or P2A (e.g., Porcine teschovirus-12A).

[0437] (iii) Origin of replication To propagate the vector in a host cell, the vector may contain one or more origin of replication sites (often referred to as "ori"), for example, a nucleic acid sequence corresponding to the oriP of EBV as described above, or an engineered oriP with a similar or enhanced function in programming, which is a specific nucleic acid sequence at which replication is initiated. Alternatively, the origin of replication or autonomously replicating sequences (ARS) of other extrachromosomally replicating viruses as described above can be used.

[0438] B. Selectable and Screenable Markers In some embodiments, cells containing the constructs of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector. Such a marker confers a distinguishable change to the cell, allowing easy identification of cells containing the expression vector. Generally, a selection marker confers a property that allows for selection. A positive selection marker is one whose presence allows for its selection, and a negative selection marker is one whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0439] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of conditions, other types of markers are contemplated, including screenable markers such as GFP, whose basis is colorimetric analysis. Alternatively, screenable enzymes as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT), may be utilized. Those skilled in the art will also know how to use immunological markers, possibly in combination with FACS analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screenable markers are well known to those skilled in the art.

[0440] 2. Other nucleic acid delivery methods In addition to viral delivery of nucleic acids encoding antigen receptors, the following are additional methods of recombinant gene delivery to a given cell (eg, NK cell) and thus are contemplated in this disclosure.

[0441] Introduction of nucleic acids, such as DNA or RNA, into immune cells of the present disclosure may use any suitable method for nucleic acid delivery for cell transformation, as described herein or known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA by, for example, ex vivo transfection, injection, including microinjection, electroporation, calcium phosphate precipitation, use of DEAE dextran followed by polyethylene glycol, direct sonic loading, liposome-mediated transfection and receptor-mediated transfection, microprojectile bombardment, agitation with silicon carbide fibers, Agrobacterium-mediated transformation, desiccation / inhibition-mediated DNA uptake, and any combination of such methods. By applying techniques such as these, organelles, cells, tissues, or organisms may be stably or transiently transformed.

[0442] A. Transposon-Based Modification Methods In general, gene transfer systems may include transposon-based or viral-based integration systems.

[0443] In some embodiments, the gene transfer system comprises a transposon system. DNA transposons can translocate via a non-replicative "cut and paste" mechanism. This mechanism requires the recognition of two inverted terminal repeats (ITRs) by a catalytic enzyme, i.e., a transposase, which can cleave the target and thus release the DNA transposon from the donor template. Upon excision, the DNA transposon may then be integrated into an acceptor DNA that is cleaved by the same transposase. In some natural structures of DNA transposons, the DNA transposon is flanked by two ITRs and may contain a gene that codes for a transposase that catalyzes transposition.

[0444] The transposon system offers many advantages for the integration of nucleic acids, for example, compared to viral vectors. For example, transposons can carry larger cargo, which may be advantageous for delivering one or more of the CARs, functional effector elements, and / or cytokines disclosed herein to immune cells (e.g., NK cells). In addition, transposons can include, for example, CRISPR tools (e.g., with cargo), which allows for multiple manipulation of cells.

[0445] The transposon system comprises (i) a plasmid backbone with inverted terminal repeats (ITRs) and (ii) a transposase enzyme that recognizes the ITRs. The terms "inverse terminal repeat", "inverted terminal repeat", or "ITR" are used interchangeably herein and refer to short sequence repeats that flank the transposase gene in natural transposons or the cargo polynucleotide sequence in artificially engineered transposons. Two inverted terminal repeats are generally required for mobilization of a transposon in the presence of a corresponding transposase. The inverted terminal repeats described herein may comprise one or more direct repeat (DR) sequences. These DR sequences are usually embedded in the inverted terminal repeats (ITRs) of the element. The compositions and methods of the present disclosure, in various embodiments, comprise one or more artificially engineered transposons. The engineered transposon may comprise an ITR, a cargo nucleotide sequence, or a cargo cassette. "Cargo cassette" as used herein in relation to a transposon refers to a nucleotide sequence that includes a left ITR at the 5' end and a right ITR at the 3' end, and a nucleotide sequence located between the left and right ITRs. The nucleotide sequences flanking the ITRs are nucleotide sequences intended for integration into an acceptor DNA. The cargo cassette may in some embodiments be included in a vector, such as a plasmid. A "cargo nucleotide sequence" is a nucleotide sequence flanked at each end by ITRs (e.g., a nucleotide sequence intended for integration into an acceptor DNA), where the ITRs are heterologous to the nucleotide sequence. The cargo cassette may be engineered artificially.

[0446] Transposons and Transposases

[0447] Exemplary transposon systems for use as described in this disclosure include piggyBac, hyperactive piggyBac, Sleeping Beauty (SB), hyperactive Sleeping Beauty (SB100x), SB11, SB110, Tn7, TcBuster, hyperactive TcBuster, Frog Prince, IS5, TnlO, Tn903, SPIN, hAT, Hermes, Hobo, AeBusterl, AeBuster2, AeBuster3, BtBusterl, BtBuster2, CfBusterl, CfBuster2, Tol2, mini-Tol2, Tc3, Mos1, MuA, Himar I, Helitron, and engineered forms of transposase family enzymes (Zhang et al. (2009) PLoS Genet. 5:e1000689; Wilson et al. (2007) J. Microbiol. Methods, 2009). 71:332-5, the contents of which are incorporated herein by reference in their entirety. Exemplary transposons also include the transposons described in Arensburger et al. (2011) Genetics 188(1):45-57, the contents of which are incorporated herein by reference in their entirety, or the SPACE INVADERS (SPIN) transposons (e.g., Pace et al. (2008) Proc. Natl. Acad. Sci. USA. 2008;105(44):17023-17028, the contents of which are incorporated herein by reference in their entirety).

[0448] In some embodiments, the gene transfer system can be delivered to the cell as DNA-encoded, mRNA-encoded, protein, or nucleoprotein complex. Alternatively, the gene transfer system can be integrated into the genome of the host cell using, for example, retrotransposons, random plasmid integration, recombinase-mediated integration, homologous recombination-mediated integration, or non-homologous end joining-mediated integration.Further examples of transposon systems that can be used in certain embodiments of the compositions and methods provided herein include Staphylococcus aureus Tn552 (Colegio et al, J. BacterioL, 183:2384-8, 2001; Kirby C et al, Mol. Microbiol, 43:173-86, 2002), Tyl (Devine & Boeke, Nucleic Acids Res., 22:3765-72, 1994 and International Publication WO 95 / 23875), transposon Tn7 (Craig, NL, Science. 271:1512, 1996; Craig, NL, Review in: Curr Top Microbiol Immunol, 204:27-48, 1996), Tn / O and IS10 (Kleckner N, et al, Curr Top Microbiol Immunol, 204:27-48, 1996). Immunol, 204:49-82, 1996), Mariner transposase (Lampe D J, et al, EMBO J., 15:5470-9, 1996), Tel (Plasterk RH, Curr. Topics Microbiol. Immunol, 204:125-43, 1996), P Element (Gloor, GB, Methods Mol. Biol, 260:97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265:18829-32, 1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204:1-26, 1996), retroviruses (Brown, et al, Proc Natl Acad Sci USA, 86:2525-9, 1989), as well as yeast retrotransposons (Boeke & Corces, Annu Rev Microbiol. 43:403-34, 1989). The entire contents of each of the foregoing references are incorporated herein by reference.

[0449] Transposition efficiency can be measured by the percentage of successful transposition events that occurred in a population of host cells, normalized by the amount of transposon and transposase introduced into the population of host cells. Often, when comparing the transposition efficiency of two or more transposases, the same transposon construct is paired with each of the two or more transposases for transfection of host cells under the same or similar transfection conditions. The amount of transposition events in a host cell can be examined by various approaches. For example, a transposon construct can be designed to include a reporter gene located between the inverted repeats, and transfected cells that are positive for the reporter gene can be counted as cells in which successful transposition events have occurred, thereby providing an estimate of the amount of transposon transposition events. Another non-limiting example includes sequencing the host cell genome to examine the insertion of the cassette cargo of the transposon. In some embodiments, when comparing the transposition efficiency of two or more different transposons, the same transposase can be paired with each of the different transposons for transfection of host cells under the same or similar transfection conditions. Similar approaches as described above and other methods commonly known to those skilled in the art can also be performed to compare transposition efficiencies.

[0450] Polynucleotides encoding the transposase system

[0451] One aspect of the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding a transposase described herein. In some embodiments, the polynucleotide further comprises a nucleotide sequence of a transposon (e.g., an engineered transposon) recognizable by the transposase. In some embodiments, the polynucleotide is comprised in an expression vector. In some embodiments, the expression vector is a DNA plasmid. In some embodiments, the expression vector is a minicircle vector. In some embodiments, the expression vector is a nanoplasmid.

[0452] As used herein, the term "minicircle vector" may refer to small circular plasmid derivatives that contain most, if not all, of the prokaryotic vector parts (e.g., control sequences or non-functional sequences of prokaryotic origin).

[0453] For transposon-based genome editing applications, in some embodiments, it may be desirable to design the transposon for use in a binary system based on two separate plasmids, whereby the nucleic acid sequence encoding the transposase is physically separated from the transposon nucleic acid sequence containing the gene of interest flanked by inverted repeat sequences. Co-delivery of the transposon and transposase-encoding plasmids to the target cell allows transposition by a traditional cut-and-paste mechanism. In some other embodiments, the transposon-based system described herein may include a polynucleotide that includes both the nucleic acid sequence encoding the transposase described herein and the nucleic acid sequence of the transposon described herein, i.e., the transposase and transposon nucleic acids are present in the same plasmid.

[0454] One of the limitations of the application of plasmid vectors is that promoter inactivation mediated by the bacterial region of the vector (i.e., the region encoding the bacterial replication origin and the selection marker) shortens the expression period of the transgene from the plasmid vector (Chen et al., 2004. Gene Ther 11:856-864; Suzuki et al., 2006. J Virol 80:3293-3300). This results in a short period of transgene expression. A strategy to improve the expression period of the transgene is to remove the bacterial region of the plasmid. For example, minicircle vectors that do not contain the bacterial region have been developed. By removing the bacterial region in the minicircle vector, the expression period of the transgene has been improved (Chen et al., 2004). In the minicircle vector, the polyadenylation signal of the eukaryotic region is covalently linked to the promoter of the eukaryotic region via a short spacer, typically less than 200 bp, composed of recombination binding sites. This linkage (spacer region) can tolerate much longer spacer sequences, because long spacers over 1 kb in length caused silencing of transgene expression in vivo, while short spacers less than 500 bp showed transgene expression patterns similar to conventional minicircle DNA vectors (Lu et al., 2012. Mol Ther. 20:2111-9).

[0455] In some embodiments, the vector useful in various aspects of the present disclosure is a nanoplasmid vector. As used herein, the term "nanoplasmid vector" refers to a vector that combines an RNA selection marker with an R6K, ColE2, or ColE2-related origin of replication. The nanoplasmid vector can be selected from the nanoplasmid vectors disclosed in any of International PCT Publication Nos. 2014 / 035457, 2014 / 077866, and 2019 / 183248, each of which is incorporated herein by reference in its entirety. For example, International PCT Publication No. 2014 / 035457 discloses a minimized nanoplasmid vector that utilizes RNA-OUT antibiotic-free selection and replaces the large 1000bp pUC origin of replication with a novel 300bp R6K origin, which improves expression from the plasmid. By reducing the spacer region linking the 5' and 3' ends of the transgene expression cassette with the R6K origin-RNA-OUT backbone to less than R500bp, expression times were improved to that of conventional minicircle DNA vectors. The 1.1 kbp FAR4 vector pUC origin tRNA antibiotic-free selection spacer improved expression times compared to the 2.2 kb pUC origin kanR antibiotic selection marker spacer region (Quiviger et al., 2014. Gene Therapy 21:1001-1007). This shows that improved expression times can be obtained with several bacterial regions up to 1.1 kb. Improved expression levels compared to plasmid vectors are also observed with several spacer regions less than 1.1 kb. For example, pVAX1 derivatives with a reduced 2 kb bacterial backbone to 1.2, 1.1, or 0.7 kb show more than 2-fold expression improvement compared to the parent pVAX1 vector. NTC8685 derivatives (nanoplasmid vectors) in which the 1.5 kb bacterial backbone has been reduced to 0.9 kb, 466 bp, or 281 bp, show greater than 2-fold improved expression compared to the parent NTC8685 vector.

[0456] In some embodiments, the nanoplasmid vectors are useful for viral and non-viral gene therapy, viral and non-viral cell therapy, and more specifically for improving viral and non-viral vector manufacturing yield and quality, reducing transfection-associated toxicity, improving transposition from non-viral transposon vectors, improving packaging titers from viral vectors, improving expression of transgenes encoded in viral and non-viral vectors, and eliminating antibiotic resistance marker gene transfer by viral and non-viral vectors, as described in PCT International Publication No. WO2019 / 183248, which is incorporated by reference herein in its entirety.

[0457] In some embodiments, the Nano plasmid vector comprises a modification that improves replication of the vector. In some embodiments, the Nano plasmid vector replicates utilizing a PolIII dependent origin of replication. In some embodiments, the Nano plasmid vector replicates utilizing a PolI dependent origin of replication. In some embodiments, the Nano plasmid vector comprises an antibiotic selection marker. In some embodiments, the Nano plasmid vector does not comprise an antibiotic selection marker. In some embodiments, the Nano plasmid vector comprises an RNA selection marker.

[0458] B. Other Modification Methods In some embodiments of the methods of the present disclosure, the modified immune cells of the present disclosure may be produced by introducing a transgene into the immune cells of the present disclosure. The introducing step may include delivery of a nucleic acid sequence and / or a genome editing construct via a non-translocation delivery system.

[0459] In some embodiments of the methods of the present disclosure, introducing the nucleic acid sequence and / or genome editing construct into immune cells ex vivo, in vivo, in vitro, or in situ includes one or more of local delivery, adsorption, absorption, electroporation, spinfection, co-culture, transfection, mechanical delivery, ultrasonic delivery, vibration delivery, magnetofection, or nanoparticle-mediated delivery. In some embodiments of the methods of the present disclosure, introducing the nucleic acid sequence and / or genome editing construct into immune cells ex vivo, in vivo, in vitro, or in situ includes liposome transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In some embodiments of the methods of the present disclosure, introducing the nucleic acid sequence and / or genome editing construct into immune cells ex vivo, in vivo, in vitro, or in situ by mechanical transfection includes cell squeezing, cell bombardment, or gene gun technology. In some embodiments of the methods of the present disclosure, introducing nucleic acid sequences and / or genome editing constructs into immune cells ex vivo, in vivo, in vitro, or in situ by nanoparticle-mediated transfection includes liposome delivery, micelle delivery, and polymersome delivery.

[0460] In some embodiments of the method of the present disclosure, the ex vivo, in vivo, in vitro, or in situ introduction of the nucleic acid sequence and / or genome editing construct into the immune cell includes a non-viral vector. In some embodiments, the non-viral vector includes a nucleic acid. In some embodiments, the non-viral vector includes a plasmid DNA, a linear double-stranded DNA (dsDNA), a linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmid, minicircle DNA, a single-stranded oligodeoxynucleotide (ssODN), a DDNA oligonucleotide, a single-stranded mRNA (ssRNA), and a double-stranded mRNA (dsRNA). In some embodiments, the non-viral vector includes a transposon of the present disclosure.

[0461] In some embodiments of the disclosed methods, an enzyme may be used to generate a strand break in the host genome to facilitate delivery or integration of the transgene. In some embodiments, the enzyme generates a single-strand break. In some embodiments, the enzyme generates a double-strand break. In some embodiments, examples of cleavage-inducing enzymes include, but are not limited to, transposases, integrases, endonucleases, meganucleases, megaTALs, CRISPR-Cas9, CRISPR-CasX, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs). In some embodiments, the cleavage-inducing enzymes can be DNA-encoded, mRNA-encoded, delivered to cells as proteins, or as nucleoprotein complexes with guide RNAs (gRNAs).

[0462] In some embodiments of the methods of the present disclosure, site-specific transgene integration is controlled by vector-mediated integration site bias. In some embodiments, vector-mediated integration site bias is controlled by a selected lentiviral vector. In some embodiments, vector-mediated integration site bias is controlled by a selected gamma retroviral vector.

[0463] In some embodiments of the methods of the present disclosure, the site-specific transgene integration site is a non-stable chromosomal insertion. In some embodiments, the integrated transgene may be silenced, removed, excised, or further modified.

[0464] In some embodiments of the methods of the present disclosure, the genomic modification is a non-stable integration of the transgene. In some embodiments, the non-stable integration can be a transient non-chromosomal integration, a semi-stable non-chromosomal integration, a semi-persistent non-chromosomal insertion, or a non-stable chromosomal insertion. In some embodiments, the transient non-chromosomal insertion can be epi-chromosomal or cytoplasmic.

[0465] In some embodiments, the transient non-chromosomal insertion of the transgene does not integrate into a chromosome and the modified genetic material is not replicated during cell division.

[0466] In some embodiments of the disclosed methods, the genomic modification is semi-stable or persistent non-chromosomal integration of the transgene. In some embodiments, the DNA vector encodes a scaffold / matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of the non-viral vector, allowing autonomous replication in the nuclei of dividing cells.

[0467] In some embodiments of the methods of the present disclosure, the genomic modification is a non-stable chromosomal integration of the transgene. In some embodiments, the integrated transgene may be silenced, removed, excised, or further modified.

[0468] In some embodiments of the disclosed method, the modification of the genome by the introduction of a transgene can occur by host cell-directed double-strand break repair (homology-directed repair) by homologous recombination (HR), microhomology-mediated end joining (MMEJ), non-homologous end joining (NHEJ), transposase enzyme-mediated modification, integrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or recombinase enzyme-mediated modification. In some embodiments, the modification of the genome by the introduction of a transgene can occur via CRISPR-Cas9, CRISPR-CasX, TALEN, or ZFN.

[0469] C. Nanoparticle Delivery Poly(histidine) (i.e., poly(L-histidine)) is a pH-sensitive polymer because the imidazole ring provides a lone pair of electrons on the unsaturated nitrogen. That is, poly(histidine) has amphoteric properties due to protonation-deprotonation. Various embodiments enable intracellular delivery of gene editing tools by complexing with poly(histidine)-based micelles. In particular, various embodiments provide triblock copolymers consisting of a hydrophilic block, a hydrophobic block, and a charged block. In some embodiments, the hydrophilic block may be poly(ethylene oxide) (PEO) and the charged block may be poly(L-histidine). An example of a triblock copolymer that may be used in various embodiments is PEO-b-PLA-b-PHIS, where the number of repeat units in each block varies by design. Gene editing tools may be various molecules recognized to be capable of modifying, repairing, adding, and / or silencing genes in various cells. Accurate and efficient repair of DNA double-strand breaks (DSBs) is important for maintaining genome stability in cells. Structural damage to DNA can occur randomly and unpredictably in the genome either by a number of intracellular factors (e.g., nucleases, reactive oxygen species, etc.) and external forces (e.g., ionizing radiation, ultraviolet (UV) radiation, etc.). In particular, accurate and efficient repair of DNA double-strand breaks (DSBs) is important for maintaining genome stability. Thus, cells are naturally equipped with many DNA repair mechanisms that can be utilized to modify DNA sequences with controlled DSBs at specific sites. Thus, genetic modification tools may consist of programmable sequence-specific DNA binding modules associated with non-specific DNA nucleases that introduce DSBs into the genome. For example, CRISPRs are genetic loci found primarily in bacteria that contain short direct repeats and are part of an acquired prokaryotic immune system that confers resistance to exogenous sequences such as plasmids and phages. RNA-guided endonucleases are programmable genetic engineering tools adapted from the CRISPR / CRISPR-associated protein 9 (Cas9) system, a component of prokaryotic innate immunity.

[0470] Diblock copolymers that can be used as intermediates to make the triblock copolymers of the micelles of the embodiments may have hydrophilic biocompatible poly(ethylene oxide) (PEO), which is chemically synonymous with PEG, conjugated to a variety of hydrophobic aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(orthoesters), poly(peptides), poly(phosphazenes), and poly(saccharides), including but not limited to poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and poly(trimethylene carbonate) (PTMC). Polymeric micelles composed of 100% PEGylated surfaces have improved in vitro chemical stability, increased in vivo bioavailability, and extended blood circulation half-life. For example, the aliphatic polyesters that make up the membrane portion of the polymeric micelles are degraded by hydrolysis of their ester bonds under physiological conditions, such as the human body. Due to their biodegradable nature, aliphatic polyesters have attracted considerable attention for use as implantable biomaterials in drug delivery devices, bioabsorbable sutures, adhesion barriers, and scaffolds for tissue engineered wound repair.

[0471] In various embodiments, the molecules required for gene editing (i.e., gene editing tools) may be delivered to cells using one or more micelles formed from self-assembling triblock copolymers containing poly(histidine). As used herein, the term "gene editing" refers to the insertion, deletion, or replacement of nucleic acids in genomic DNA to add, destroy, or modify the function of the product encoded by the gene. Various gene editing systems require at least the introduction of a cleavage enzyme (such as a nuclease or recombinase) that cleaves genomic DNA to destroy or activate gene function.

[0472] Furthermore, gene editing systems involving the insertion of new or existing nucleotides / nucleic acids require the delivery of an insertion tool (e.g., DNA template vector, transposable element (transposon or retrotransposon)) to the cell in addition to a cutting enzyme (e.g., nuclease, recombinase, integrase, or transposase). Examples of such insertion tools for recombinases may include DNA vectors. Other gene editing systems require the delivery of an integrase with an insertion vector, a transposase with a transposon / retrotransposon, and the like. In some embodiments, an example of a recombinase that may be used as a cutting enzyme is CRE recombinase. In various embodiments, examples of integrases that may be used in the insertion tool include viral-based enzymes taken from any of a number of viruses, including but not limited to AAV, gamma retroviruses, and lentiviruses. Examples of transposons / retrotransposons that may be used in the insertion tool include, but are not limited to, piggyBac® transposon, Sleeping Beauty transposon, TcBuster transposon, and L1 retrotransposon.

[0473] In certain embodiments of the disclosed method, the transgene is delivered in vivo. In certain embodiments of the disclosed method, the in vivo transgene delivery can occur by topical delivery, adsorption, absorption, electroporation, spinfection, co-culture, transfection, mechanical delivery, ultrasound delivery, vibration delivery, magnatofection, or nanoparticle-mediated delivery. In certain embodiments of the disclosed method, the in vivo transgene delivery by transfection can occur by liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In certain embodiments of the disclosed method, the in vivo mechanical transgene delivery can occur by cell squeezing, cell bombardment, and gene gun. In certain embodiments of the disclosed method, the in vivo nanoparticle-mediated transgene delivery can occur by liposomal delivery, micelle delivery, and polymersome delivery. In various embodiments, nucleases that can be used as cleavage enzymes include, but are not limited to, Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases.

[0474] In various embodiments, the gene editing system described herein, particularly proteins and / or nucleic acids, may be complexed with nanoparticles that are poly(histidine)-based micelles. In particular, at certain pHs, poly(histidine)-containing triblock copolymers assemble to form micelles with positively charged poly(histidine) units on the surface, which allows them to complex with negatively charged gene editing molecules. These nanoparticles can be used to bind and release proteins and / or nucleic acids in a pH-dependent manner, providing an efficient and selective mechanism for making the desired genetic modifications. In particular, this micelle-based delivery system offers substantial flexibility with respect to charged materials, as well as large payload capacity and targeted release of nanoparticle payloads. In one example, site-specific cleavage of double-stranded DNA may be enabled by delivery of nucleases using poly(histidine)-based micelles.

[0475] Various embodiments enable intracellular delivery of gene editing tools by complexing with poly(histidine)-based micelles. In particular, various embodiments provide triblock copolymers consisting of a hydrophilic block, a hydrophobic block, and a charged block. In some embodiments, the hydrophilic block can be poly(ethylene oxide) (PEO) and the charged block can be poly(L-histidine). An example of a triblock copolymer that can be used in various embodiments is PEO-b-PLA-b-PHIS, where the number of repeat units in each block varies by design. Without being bound by any particular theory, it is believed that in the micelles formed by the triblock copolymers of various embodiments, the hydrophobic blocks aggregate to form a core, leaving the hydrophilic and poly(histidine) blocks at the ends to form one or more surrounding layers.

[0476] In certain embodiments of the method of the present disclosure, a non-viral vector is used for transgene delivery. In certain embodiments, the non-viral vector is a nucleic acid. In certain embodiments, the nucleic acid non-viral vector is a plasmid DNA, a linear double-stranded DNA (dsDNA), a linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmid, minicircle DNA, a single-stranded oligodeoxynucleotide (ssODN), a DDNA oligonucleotide, a single-stranded mRNA (ssRNA), and a double-stranded mRNA (dsRNA). In certain embodiments, the non-viral vector is a transposon. In certain embodiments, the transposon is TcBuster.

[0477] In certain embodiments of the disclosed method, transgene delivery may occur via a viral vector. In certain embodiments, the viral vector is a non-integrating non-chromosomal vector. Non-integrating non-chromosomal vectors may include adeno-associated virus (AAV), adenovirus, and herpes virus. In certain embodiments, the viral vector is an integrating chromosomal vector. Inintegrating chromosomal vectors may include adeno-associated vector (AAV), lentivirus, gamma retrovirus.

[0478] In certain embodiments of the disclosed method, the delivery of the transgene may occur by a combination of vectors. Exemplary but non-limiting vector combinations may include a viral vector and a non-viral vector, two or more non-viral vectors, or two or more viral vectors. Exemplary but non-limiting vector combinations may include a DNA-derived vector and an RNA-derived vector, an RNA transcriptase and a reverse transcriptase, a transposon and a transposase, a non-viral vector and an endonuclease, and a viral vector and an endonuclease.

[0479] In certain embodiments of the methods of the present disclosure, the genomic modification can be stable integration of the transgene, transient integration of the transgene, site-specific integration of the transgene, or biased integration of the transgene.

[0480] In certain embodiments of the disclosed methods, the genome modification may be stable chromosomal integration of the transgene. In certain embodiments, the stable chromosomal integration may be random integration, site-specific integration, or biased integration. In certain embodiments, the site-specific integration may be non-assisted or assisted. In certain embodiments, the assisted site-specific integration is co-delivered with a site-specific nuclease. In certain embodiments, the site-specific nuclease comprises a transgene with 5' and 3' nucleotide sequence extensions that contain homology to the upstream and downstream regions of the genome integration site. In certain embodiments, the transgene with homologous nucleotide extensions allows for genome integration by homologous recombination, microhomology-mediated end joining, or non-homologous end joining. In certain embodiments, the site-specific integration occurs at a safe harbor site. A genomic safe harbor site can provide for the integration of new genetic material in a manner that ensures that the newly inserted genetic element is functional (e.g., expressed at a therapeutically effective expression level) and does not cause deleterious changes to the host genome that pose a risk to the host organism. Potential genomic safe harbors include intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), the naturally occurring integration site of the AAV virus on chromosome 19, the chemokine (CC motif) receptor 5 (CCR5) gene site, and the human ortholog of the mouse Rosa26 locus.

[0481] In certain embodiments, site-specific transgene integration occurs at a site that disrupts expression of a target gene. In certain embodiments, disruption of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. In certain embodiments, exemplary target genes targeted by site-specific integration include, but are not limited to, any immunosuppressive genes and genes involved in allogeneic rejection.

[0482] In certain embodiments, site-specific transgene integration occurs at a site that results in enhanced expression of the target gene, hi certain embodiments, enhanced target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0483] In certain embodiments of the disclosed method, an enzyme may be used to generate a strand break in the host genome to facilitate delivery or integration of the transgene. In certain embodiments, the enzyme generates a single-strand break. In certain embodiments, the enzyme generates a double-strand break. In certain embodiments, examples of cleavage-inducing enzymes include, but are not limited to, transposases, integrases, endonucleases, meganucleases, megaTALs, CRISPR-Cas9, CRISPR-CasX, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). In certain embodiments, the cleavage-inducing enzymes may be delivered to cells as DNA-encoded, mRNA-encoded, proteins, or nucleoprotein complexes with guide RNAs (gRNAs).

[0484] In certain embodiments of the disclosed methods, site-specific transgene integration is controlled by vector-mediated integration site bias. In certain embodiments, vector-mediated integration site bias is controlled by a selected lentiviral vector. In certain embodiments, vector-mediated integration site bias is controlled by a selected gamma retroviral vector.

[0485] In certain embodiments of the disclosed method, the site-specific transgene integration site is a non-stable chromosomal insertion. In certain embodiments, the integrated transgene may be silenced, removed, excised, or further modified. In certain embodiments of the disclosed method, the genome modification is a non-stable integration of the transgene. In certain embodiments, the non-stable integration may be a transient non-chromosomal integration, a semi-stable non-chromosomal integration, a semi-permanent non-chromosomal insertion, or a non-stable chromosomal insertion. In certain embodiments, the transient non-chromosomal insertion may be epi-chromosomal or cytoplasmic. In certain embodiments, the transient non-chromosomal insertion of the transgene is not integrated into a chromosome, and the modified genetic material is not replicated during cell division.

[0486] In certain embodiments of the disclosed methods, the genomic modification is semi-stable or persistent non-chromosomal integration of the transgene. In certain embodiments, the DNA vector encodes a scaffold / matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of the non-viral vector, allowing autonomous replication in the nuclei of dividing cells.

[0487] In certain embodiments of the disclosed methods, the genomic modification is non-stable chromosomal integration of the transgene. In certain embodiments, the integrated transgene may be silenced, removed, excised, or further modified.

[0488] In certain embodiments of the method of the present disclosure, the modification of genome by transgene insertion can occur by host cell-directed double-strand break repair (homology-directed repair) by homologous recombination (HR), microhomology-mediated end joining (MMEJ), non-homologous end joining (NHEJ), transposase enzyme-mediated modification, integrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or recombinase enzyme-mediated modification. In certain embodiments, the modification of genome by transgene insertion can occur via CRISPR-Cas9, CRISPR-CasX, TALEN, or ZFN.

[0489] In certain embodiments of the disclosed methods, the cells with in vivo or ex vivo genomic modifications can be germline or somatic cells. In certain embodiments, the modified cells can be human, non-human, mammalian, rat, mouse, or canine cells. In certain embodiments, the modified cells can be differentiated, undifferentiated, or immortalized. In certain embodiments, the modified undifferentiated cells can be stem cells. In certain embodiments, the modified cells can be differentiated, undifferentiated, or immortalized. In certain embodiments, the modified undifferentiated cells can be induced pluripotent stem cells. In certain embodiments, the modified cells can be T cells, hematopoietic stem cells, natural killer cells, macrophages, dendritic cells, monocytes, megakaryocytes, or osteoclasts. In certain embodiments, the modified cells can be modified while the cells are in a quiescent, activated, resting, interphase, prophase, metaphase, anaphase, or telophase. In certain embodiments, the modified cells can be fresh, cryopreserved, bulk, and sorted into subpopulations derived from whole blood, leukapheresis, or from immortalized cell lines.

[0490] B. ZFPs and ZFNs In some embodiments, the DNA targeting molecule includes a DNA binding protein, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), fused to an effector protein, such as an endonuclease. Examples include ZFNs, TALEs, and TALENs.

[0491] In some embodiments, the DNA targeting molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain (a region of amino acid sequence within a binding domain whose structure is stabilized by the coordination of a zinc ion) within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial ZFP domains that target specific DNA sequences, typically 9-18 nucleotides long, generated by the assembly of individual fingers.

[0492] ZFPs include those with two, three, four, five, or six fingers, in which the single finger domain is about 30 amino acids long and contains an alpha helix containing two invariant histidine residues coordinated through two cysteines and zinc in a single beta turn. In general, the sequence specificity of a ZFP can be altered by making amino acid substitutions at the four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is not naturally occurring and is engineered to bind, for example, to a selected target site.

[0493] In some embodiments, the DNA target molecule is or comprises a zinc finger DNA binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN). In some embodiments, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one liS-type restriction enzyme, which may or may not be engineered, and one or more zinc finger binding domains. In some embodiments, the cleavage domain is derived from the liS-type restriction endonuclease FokI. FokI catalyzes double-stranded cleavage of DNA, typically 9 nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the other strand.

[0494] Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) has partnered with Sigma-Aldrich (St. Lewis, MO, USA) to develop a platform for zinc finger construction (CompoZr) that allows researchers to bypass the construction and validation of all-in-one zinc fingers and provides specifically targeted zinc fingers for thousands of proteins (Gaj et al, Trends in Biotechnology, 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers are used or custom designed. (See, for example, Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTil-lKT, and PZD0020).

[0495] C. TAL, TALE, and TALEN In some embodiments, the DNA targeting molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as a transcription activator-like protein effector (TALE) protein. See, e.g., U.S. Patent Application Publication No. 2011 / 0301073, which is incorporated herein by reference in its entirety.

[0496] A TALE DNA binding domain or TALE is a polypeptide that contains one or more TALE repeat domains / units. The repeat domain is responsible for TALE binding to the cognate target DNA sequence. A single "repeat unit" (also called "repeat") is typically 33-35 amino acids in length and shows at least some sequence homology to other TALE repeat sequences in naturally occurring TALE proteins. Each TALE repeat unit contains one or two DNA binding residues that constitute a repeat variable dipeptide (RVD), typically at positions 12 and / or 13 of the repeat. The natural (canonical) codes for DNA recognition of these TALEs have been determined, such that the HD sequence at positions 12 and 13 binds to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NO binds to T, and non-canonical (atypical) RVDs are also known. In some embodiments, TALEs may be targeted to any gene by design of a TAL array with specificity for the target DNA sequence. The target sequence generally starts with a thymidine.

[0497] In some embodiments, the molecule is a DNA-binding endonuclease, such as a TALE nuclease (TALEN). In some aspects, a TALEN is a fusion protein that includes a DNA-binding domain from a TALE and a nuclease catalytic domain for cleaving a nucleic acid target sequence.

[0498] In some embodiments, TALENs recognize and cleave target sequences in genes. In some aspects, DNA cleavage results in double-strand breaks. In some aspects, cleavage promotes the rate of homologous recombination or non-homologous end joining (NHEJ). In general, NHEJ is an imperfect repair process that often results in alteration of the DNA sequence at the cleavage site. In some aspects, the repair mechanism involves rejoining what remains of the two DNA ends by direct religation or so-called microhomology-mediated end joining. In some embodiments, repair by NHEJ produces small insertions or deletions that can be used to disrupt and thereby suppress genes. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells that have undergone a cleavage-induced mutagenesis event, i.e., a mutagenesis event following an NHEJ event, can be identified and / or selected by methods well known in the art.

[0499] In some embodiments, TALE repeats are assembled to specifically target genes. (Gaj et al., 2013). A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., 2013). Custom-designed TALE arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, Kentucky, USA), and Life Technologies (Grand Island, New York, USA). In particular, TALENs targeting CD38 are commercially available (see Gencopoeia, Cat. Nos. HTN222870-1, HTN222870-2, and HTN222870-3). Exemplary molecules are described, for example, in US Patent Application Publication Nos. 2014 / 0120622 and 2013 / 0315884.

[0500] In some embodiments, the TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some aspects, the plasmid vectors can include a selection marker that provides for identification and / or selection of cells that have received the vector.

[0501] D. Meganucleases and MegaTAL In certain embodiments, the nuclease comprises a meganuclease (homing endonuclease) or a portion thereof that exhibits cleavage activity. In some embodiments, "meganuclease", also referred to as "homing endonuclease", refers to an endo-deoxyribonuclease characterized by a large recognition site (double-stranded DNA sequence of about 12 to about 40 base pairs). Naturally occurring meganucleases recognize cleavage sites of 15 to 40 base pairs and are generally classified into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family, and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. Their recognition sequences are known. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue.

[0502] Naturally occurring meganucleases, primarily DNA-binding domains from the LAGLIDADG family, have been used to facilitate site-specific genome modification in plants, yeast, Drosophila, mammalian cells, and mice, but this approach relies on the modification of either homologous genes that preserve the meganuclease recognition sequence (Monet et al. (1999), Biochem. Biophysics. Res. Common. 255:88-93), or on previously engineered genomes in which the recognition sequence has been introduced (Route et al. (1994), Mol. Cell. Biol. 14:8096-106; Chilton et al. (2003), Plant Physiology. 133:956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93:5055-60; Rong et al. (2002), Genes Dev. 16:1568-81; Gouble et al. (2003), Genes Dev. 16:1568-81; Gouble et al. (2003), Genes Dev. 16:1568-81). al. (2006), J. Gene Med. 8(5):616-622). Therefore, attempts have been made to engineer meganucleases to exhibit novel binding specificities at medically or biotechnologically relevant sites (Porteus et al. (2005), Nat. Biotechnol. 23:967-73; Sussman et al. (2004), J. Mol. Biol. 342:31-41; Epinat et al. (2003), Nucleic Acids Res. 31:2952-62; Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Current Genes 441:656-659). Therapy 7:49-66; U.S. Patent Application Publication Nos. 20070117128; 20060206949; 20060153826; 20060078552; and 20040002092).Furthermore, a naturally occurring or engineered DNA binding domain from a meganuclease can be operably linked to a cleavage domain from a heterologous nuclease (e.g., FokI) and / or a cleavage domain from a meganuclease can be operably linked to a heterologous DNA binding domain (e.g., a ZFP or TALE).

[0503] In any of the nucleases described herein, the nuclease can include an engineered TALE DNA binding domain and a nuclease domain, also referred to as a TALEN (e.g., an endonuclease and / or a meganuclease domain). Methods and compositions have been published for engineering these TALEN proteins for strong site-specific interactions with user-selected target sequences (see U.S. Pat. No. 8,586,526). In some embodiments, the TALEN comprises an endonuclease (e.g., FokI) cleavage domain or cleavage half-domain. In other embodiments, the TALE nuclease is a megaTAL. These megaTAL nucleases are fusion proteins that include a TALE DNA binding domain and a meganuclease cleavage domain. The meganuclease cleavage domain is active as a monomer and does not require dimerization for activity. (See Boissel et al., (2013) Nucl Acid Res: 1-13, doi:10.1093 / nar / gkt1224). In addition, the nuclease domain may also exhibit a DNA binding function.

[0504] E.RGEN (CRISPR / Cas system) In some embodiments, the modification is performed using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonucleases (RGENs). For example, the modification can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In general, "CRISPR system" refers collectively to the transcripts and other elements involved in the expression or directing the activity of CRISPR-associated ("Cas") genes, including Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from CRISPR loci.

[0505] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system comprises a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner, and a Cas protein (e.g., Cas9) that has a nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a particular organism that contains a Type I, Type II, or Type III CRISPR system, e.g., an endogenous CRISPR system, such as Streptococcus pyogenes.

[0506] In some embodiments, a Cas nuclease and a gRNA (comprising a fusion of a target sequence-specific crRNA and a given tracrRNA) are introduced into a cell. Generally, a target site at the 5' end of the gRNA uses complementary base pairing to target the Cas nuclease to a target site, such as a gene. The target site may be selected based on its location immediately 5' to a protospacer adjacent motif (PAM) sequence, typically NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the target sequence site. Typically, a "target sequence" generally refers to a sequence to which the guide sequence is designed to have complementarity, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not required, provided there is sufficient complementarity for hybridization to occur and promote formation of a CRISPR complex.

[0507] The CRISPR system can induce a double-strand break (DSB) at the target site, followed by disruption or modification as discussed herein. In other embodiments, a Cas9 variant, considered a "nickase," is used to nick a single strand at the target site. For example, to improve specificity, a pair of nickases can be used, each of which is guided by a different pair of gRNAs targeting a sequence, simultaneously introducing a nick and introducing a 5' overhang. In other embodiments, a catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0508] The target sequence may comprise any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as in an organelle. In general, a sequence or template that can be used for recombination into a targeted locus that comprises a target sequence is referred to as an "editing template" or an "editing polynucleotide" or an "editing sequence". In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is a homologous recombination.

[0509] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence to form a complex with one or more target sequences) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence). A tracr sequence that may include or consist of all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence) may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0510] The components of the CRISPR system can be implemented in any suitable manner, meaning that the components of such systems, including the RNA-guided nuclease (e.g., Cas enzyme) and gRNA, can be delivered, formulated, or administered to a cell in any suitable form. For example, the RNA-guided nuclease can be delivered to a cell complexed with the gRNA (e.g., as a ribonucleoprotein (RNP) complex), the RNA-guided nuclease can be delivered to a cell separately from the gRNA (e.g., uncomplexed), the RNA-guided nuclease can be delivered to a cell as a polynucleotide (e.g., DNA or RNA) encoding the nuclease separate from the gRNA, or both the RNA-guided nuclease and the gRNA molecule can be delivered as polynucleotides encoding each component.

[0511] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into the cell such that expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. The components can also be delivered to the cell as ribonucleoprotein complexes, proteins, DNA, and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence can each be operably linked to another regulatory element on another vector. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector, and one or more additional vectors can provide any components of the CRISPR system not included in the first vector. The vector can include one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of the one or more vectors. Additionally, a nucleic acid encoding an endonuclease (e.g., a Cas enzyme such as Cas8 or Cas9) can be delivered together with the gRNA. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different corresponding target sequences within a cell.

[0512] The vector may include regulatory elements operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csxl2), Cas10, CasX, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0513] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector can encode a CRISPR enzyme mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide that contains the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvCI catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, the Cas9 nickase may be used in combination with a guide sequence (e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively). This combination allows both strands to be nicked and used to induce NHEJ or HDR.

[0514] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be a cell of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest by replacing at least one codon of the native sequence with a codon that is more or most frequently used in the host's genes, while maintaining the native amino acid sequence. Different species show a particular bias for the codons of a particular amino acid. Codon bias (the difference in codon usage between organisms) is often correlated with the efficiency of messenger RNA (mRNA) translation, which is believed to depend, among other things, on the properties of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The predominance of a selected tRNA in a cell generally reflects the codon that is most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be tailored for optimal gene expression in a given organism.

[0515] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50% or more, 60% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97.5% or more, or 99% or more, or more, when optimally aligned using a suitable alignment algorithm.

[0516] Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0517] CRISPR enzymes may be part of a fusion protein that includes one or more heterologous protein domains. CRISPR enzyme fusion proteins may include any additional protein sequences and, optionally, linker sequences between any two domains. Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of fusion proteins containing CRISPR enzymes are described in US20110059502, which is incorporated herein by reference.

[0518] VII. How to use In some embodiments, the present disclosure provides a method for immunotherapy comprising administering an effective amount of the immune cells of the present disclosure. In one embodiment, a medical disease or disorder is treated by transfer of an immune cell population that induces an immune response. In certain embodiments of the present disclosure, a cancer or infectious disease is treated by transfer of an immune cell population that induces an immune response. Provided herein is a method of treating or delaying the progression of cancer in an individual comprising administering to the individual an effective amount of an antigen-specific cell therapy. The methods of the present invention may be applied to the treatment of immune disorders, solid cancers, hematological cancers, and viral infections.

[0519] Tumors for which the therapeutic methods of the present invention are useful include any malignant cell type, such as those found in solid tumors or hematological tumors. In some embodiments, the cancer is a CD22-positive cancer. In some embodiments, the cancer has low expression of CD22 (e.g., a low CD22-expressing cancer). In some embodiments, the cancer is a CD19-positive cancer. In some embodiments, the cancer has low expression of CD19 (e.g., a low CD19-expressing cancer).

[0520] Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovaries, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include, but are not limited to, tumors of the bone marrow, T-cell or B-cell malignancies, myeloid malignancies, leukemia, lymphoma, blastoma, myeloma. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0521] The cancer may specifically be of the following tissue types, but is not limited to: neoplasm, malignant; tumor, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma. eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma) adenocarcinoma); noncapsular sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma Adenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia w / squamous metaplasia);thymoma, malignant;ovarian stromal tumor, malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;angiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading type Melanoma;lentigo maligna melanoma;acral lentigo melanoma;nodular melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymoma, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma;Mesonephroma, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parocytic osteosarcoma;Chondrosarcoma;Chondroblastoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Prototype stromal astrocytoma;fibrous astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;oligodendroglioma;anaplastic neuroectodermal tumor;cerebellar sarcoma;ganglioneoblastoma;neuroblastoma;retinoblastoma;olfactory neuroblastoma;meningioma, malignant;neurofibrosarcoma;schwannoma, malignant;granular cell tumor, malignant;malignant lymphoma;T-lymphoblastic leukemia;T-lymphoblastic lymphoma;Hodgkin's disease;Hodgkin's lymphoma;paragranuloma;malignant lymphoma, small lymphocytic;malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular;mycosis fungoides;other specified non-Hodgkin's lymphoma;B-cell lymphoma Lymphoma;Low-grade / follicular non-Hodgkin's lymphoma (NHL);Small lymphocytic (SL) NHL;Intermediate-grade / follicular NHL;Intermediate-grade diffuse NHL;High-grade immunoblastic NHL;High-grade lymphoblastic NHL;High-grade small noncleaved cell NHL;Giant mass disease NHL;Mantle cell lymphoma;AIDS-related lymphoma;Waldenstrom's macroglobulinemia;Malignant histiocytosis;Multiple myeloma;Mast cell sarcoma;Immunoproliferative small intestinal disease;Leukemia;Lymphocytic leukemia;Plasma cell leukemia;Erythroleukemia;Lympho-sarcoma cell myeloid leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia, acute lymphoblastic leukemia (ALL); acute lymphoblastic lymphoma; acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); myeloproliferative neoplasms; chronic myeloblastic leukemia; diffuse large cell B-cell lymphoma (DLBCL); peripheral T-cell lymphoma (PTCL); or anaplastic large cell lymphoma (ALCL).

[0522] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow and is characterized by abnormal proliferation (production by multiplication) of blood cells, usually immature white blood cells (leukocytes). It is part of a broad group of diseases called hematological neoplasms. Leukemia is a broad term that encompasses a variety of diseases. Leukemias are classified clinically and pathologically by acute and chronic forms and / or cell type of origin (myeloid or lymphoid). In some embodiments, the leukemia is a low antigen leukemia. In some embodiments, the leukemia is a low CD22 leukemia.

[0523] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need thereof, such as an individual with cancer or infectious disease. The cells then boost the individual's immune system to attack or directly attack the respective cancer cells or pathogenic cells. In some cases, the individual is provided with one or more doses of immune cells. If the individual is provided with two or more doses of immune cells, the period between doses should be sufficient to allow time for proliferation in the individual, and in certain embodiments, the period between doses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more.

[0524] Certain embodiments of the present disclosure provide a method of treating or preventing an immune-mediated disorder. 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 disease of the adrenal glands, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac polydermatitis, and the like. spate-dermatitis), chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia / fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (e.g. and membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (e.g., polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis, or dermatitis herpetiformis vasculitis), vitiligo, and Wegener's granulomatosis. Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The subject may also have an allergic disease, such as asthma.

[0525] In yet another embodiment, the subject is a recipient of a transplanted organ or stem cells, and immune cells are used to prevent and / or treat rejection. In certain embodiments, the subject has or is at risk of developing graft-versus-host disease. GVHD is a possible complication of any transplant using or involving stem cells from related or unrelated donors. There are two types of GVHD: acute and chronic. Acute GVHD occurs within the first three months after transplant. Signs of acute GVHD include a reddish skin rash on the hands and feet, which can spread with peeling and blisters of the skin and become more severe. Acute GVHD can also affect the stomach and intestines, causing muscle cramps, nausea, and diarrhea. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD is affecting the liver. Chronic GVHD is graded based on severity, with stage / grade 1 being mild and stage / grade 4 being severe. Chronic GVHD develops 3 months or more after transplantation. Symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD may affect the mucous glands of the eyes, the salivary glands in the mouth, and the glands that lubricate the stomach lining and intestines. Any of the populations of immune cells disclosed herein may be utilized. Examples of transplanted organs include solid organ transplants such as kidney, liver, skin, pancreas, lung, and / or heart, or cell transplants such as islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The transplant may be a composite transplant, such as facial tissue. The immune cells may be administered before transplantation, simultaneously with transplantation, 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 before transplantation. In one specific, non-limiting example, administration of a therapeutically effective amount of immune cells occurs 3-5 days prior to transplantation.

[0526] In some embodiments, a non-myeloablative lymphodepleting chemotherapy can be administered to the subject prior to immune cell therapy. The non-myeloablative lymphodepleting chemotherapy can be any suitable such therapy that can be administered by any suitable route. The non-myeloablative lymphodepleting chemotherapy can include, for example, administration of cyclophosphamide and fludarabine. An exemplary administration route for cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine can be administered. In certain aspects, about 60 mg / kg of cyclophosphamide is administered for two days, followed by about 25 mg / m 2 of fludarabine is administered for 5 days.

[0527] In some embodiments, the subject may be administered a non-myeloablative lymphodepleting immunotherapy prior to the immune cell therapy. The non-myeloablative lymphodepleting immunotherapy may be any suitable such therapy that may be administered by any suitable route. The non-myeloablative lymphodepleting immunotherapy may include, for example, administration of an anti-CD52 or anti-CD20 agent. In some embodiments, the lymphodepleting immunotherapy is an anti-CD52 antibody. In some embodiments, the anti-CD52 antibody is alemtuzumab. In some embodiments, the lymphodepleting immunotherapy is an anti-CD20 antibody. Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab, obinutuzumab, ibritumomab, or iodine-131 tositumomab. An exemplary route of administration of the anti-CD52 or anti-CD20 agent is intravenous. Similarly, any suitable dose of the anti-CD52 or anti-CD20 agent may be administered.

[0528] In certain embodiments, a growth factor that promotes the proliferation and activation of immune cells is administered to the subject simultaneously with the immune cells or subsequently to the immune cells. The immune cell growth factor can be any suitable growth factor that promotes the proliferation and activation of immune cells. Examples of suitable immune cell growth 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.

[0529] Therapeutically effective amounts of immune cells can be administered by a number of routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection, or infusion.

[0530] A therapeutically effective amount of immune cells for use in adoptive cell therapy is an amount that achieves a desired effect in a treated subject. For example, this may be the amount of immune cells necessary to inhibit the progression or cause the regression of an autoimmune disease or an alloimmune disease, or the amount of immune cells that can reduce symptoms such as pain and inflammation caused by an autoimmune disease. It may be the amount necessary to reduce symptoms associated with inflammation, such as pain, edema, and elevated body temperature. It may also be the am...

Claims

1. a) a first chimeric antigen receptor (CAR) comprising an antigen recognition domain that binds to a first antigen, a transmembrane domain, and an intracellular signaling domain; b) a second CAR comprising an antigen recognition domain that binds to a second antigen, a transmembrane domain, and a linker for activation of T cells (LAT) intracellular signaling domain. Genetically modified immune cells, including

2. The genetically modified immune cell of claim 1 , wherein the first antigen and the second antigen are different.

3. 2. The genetically modified immune cell of claim 1, wherein the first antigen and the second antigen are the same.

4. The genetically modified immune cell of any one of claims 1 to 3, wherein the intracellular signaling domain of the first CAR comprises a CD3 zeta intracellular signaling domain.

5. 5. The genetically modified immune cell of claim 4, wherein the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, preferably wherein the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

24.

6. The genetically modified immune cell of any one of claims 1 to 3, wherein the intracellular signaling domain of the first CAR further comprises at least one additional intracellular signaling domain selected from the group consisting of a CD97 intracellular signaling domain, a CD11a to CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8a intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, and any combination thereof.

7. 7. The genetically modified immune cell of claim 6, wherein the at least one additional intracellular signaling domain is a 4-1BB intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:

17.

8. The genetically modified immune cell of any one of claims 1 to 3, wherein the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of any one of SEQ ID NOs: 26 to 34, preferably the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO:

27.

9. 4. The genetically modified immune cell of any one of claims 1 to 3, wherein the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 26 having a lysine to arginine substitution at position 25 of SEQ ID NO: 26 (K25R), a glycine to glutamic acid substitution at position 133 of SEQ ID NO: 26 (G133E), a lysine to arginine substitution at position 206 of SEQ ID NO: 26 (K206R), or any combination of the aforementioned substitutions.

10. 4. The genetically modified immune cell of any one of claims 1 to 3, wherein the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 32 having a lysine to arginine substitution at position 25 of SEQ ID NO: 32 (K25R), a glycine to glutamic acid substitution at position 104 of SEQ ID NO: 32 (G104E), a lysine to arginine substitution at position 177 of SEQ ID NO: 32 (K177R), or any combination of the aforementioned substitutions.

11. 4. The genetically modified immune cell of any one of claims 1 to 3, wherein the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 33 having a lysine to arginine substitution at position 25 of SEQ ID NO: 33 (K25R), a glycine to glutamic acid substitution at position 103 of SEQ ID NO: 33 (G103E), a lysine to arginine substitution at position 176 of SEQ ID NO: 33 (K176R), or any combination of the aforementioned substitutions.

12. 4. The genetically modified immune cell of any one of claims 1 to 3, wherein the LAT intracellular signaling domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 34 having a lysine to arginine substitution at position 25 of SEQ ID NO: 34 (K25R), a glycine to glutamic acid substitution at position 132 of SEQ ID NO: 34 (G132E), a lysine to arginine substitution at position 205 of SEQ ID NO: 34 (K205R), or any combination of the aforementioned substitutions.

13. 4. The genetically modified immune cell of any one of claims 1 to 3, wherein the transmembrane domain of the first CAR and / or the second CAR is derived from a transmembrane domain selected from the group consisting of a CD8a transmembrane domain, a CD28 transmembrane domain, a CD3z transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, and any combination thereof.

14. 14. The genetically modified immune cell of claim 13, wherein the transmembrane domain of the first CAR is derived from a CD8α transmembrane domain comprising the amino acid sequence of SEQ ID NO:

13.

15. 14. The genetically modified immune cell of claim 13, wherein the transmembrane domain of the second CAR is derived from a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:

14.

16. The genetically modified immune cell of any one of claims 1 to 3, wherein the antigen-recognition domain of the first CAR and / or the antigen-recognition domain of the second CAR is an antibody, an antibody fragment, a single-chain antibody, a single-domain antibody, scFv, VH or VHH, or an antigen-binding fragment thereof.

17. The genetically modified immune cell of any one of claims 1 to 3, wherein the antigen-recognition domain of the first CAR and the antigen-recognition domain of the second CAR further comprise a leader domain selected from the group consisting of CD8α leader domains.

18. 18. The genetically modified immune cell of claim 17, wherein the leader domain is a CD8α leader domain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

19. The genetically modified immune cell of any one of claims 1 to 3, wherein the first antigen and the second antigen are tumor-associated antigens.

20. The tumor-associated antigen is CD19, CD22, CD20, CD138, BCMA, CD33, CD123, FLT, CLL, CD56, CD34, CD117, CD14, CD133, CD44v6, CD47, CD64, CD96, CD97, CD99, CD45, CD9, Mucl, Lewis-Y, IL1RAP, FR-beta, CD5, CD7, CD38, C 20. The genetically modified immune cell of claim 19, wherein the gene is selected from the group consisting of: D30, B7-H3, HER2, CD44v6, CEA, c-Met, EGFRvIII, Epcam, EphA2, FR-alpha, GD2, GPC3, IL13R-alpha2, IL11R-alpha, L1-CAM, mesothelin, MUC1, MUC16, NKGD2, and PSCA.

21. 21. The genetically modified immune cell of claim 20, wherein the first antigen is CD22.

22. 21. The genetically modified immune cell of claim 20, wherein the first antigen is CD19.

23. 22. The genetically modified immune cell of claim 21, wherein the second antigen is CD19.

24. 22. The genetically modified immune cell of claim 21, wherein the second antigen is CD22.

25. 23. The genetically modified immune cell of claim 22, wherein the second antigen is CD22.

26. The genetically modified immune cell of any one of claims 1 to 3, wherein the immune cell is a T cell, a natural killer (NK) cell, a natural killer (NK)-like cell, a cytokine-induced killer (CIK) cell, a hematopoietic progenitor cell, a peripheral blood (PB)-derived T cell, or an umbilical cord blood (UCB)-derived T cell.

27. 27. The genetically modified immune cell of claim 26, wherein the immune cell is a T cell.

28. The genetically modified immune cell of any one of claims 1 to 3, wherein the first CAR comprises the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 102, SEQ ID NO: 306, or SEQ ID NO:

309.

29. 4. The genetically modified immune cell of any one of claims 1 to 3, wherein the second CAR comprises the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 100, SEQ ID NO: 206, or SEQ ID NOs: 300-308.

30. 4. The genetically modified immune cell of any one of claims 1 to 3, wherein the first CAR comprises the amino acid sequence of SEQ ID NO: 102 and the second CAR comprises SEQ ID NO:

100.

31. 4. The genetically modified immune cell of any one of claims 1 to 3, wherein the first CAR comprises the amino acid sequence of SEQ ID NO: 102 and the second CAR comprises SEQ ID NO:

306.

32. 4. The genetically modified immune cell of any one of claims 1 to 3, wherein the first CAR comprises the amino acid sequence of SEQ ID NO: 309 and the second CAR comprises SEQ ID NO:

100.

33. A composition comprising the genetically modified immune cells of any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

34. 10. A composition comprising a population of cells, wherein a plurality of cells of said population comprises the genetically modified immune cell of any one of claims 1 to 3.

35. 35. The composition of claim 34, wherein the plurality of cells of the population comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage therebetween, the genetically modified immune cells of any one of claims 1-3.

36. A polynucleotide encoding the first and second CARs of the genetically modified immune cell of any one of claims 1 to 3.

37. 37. The polynucleotide of Claim 36, wherein a nucleic acid sequence encoding a self-cleaving peptide sequence is located between the nucleic acid sequence encoding the first CAR and the nucleic acid sequence encoding the second CAR.

38. 38. The polynucleotide of claim 37, wherein the self-cleaving peptide sequence comprises the amino acid sequence of SEQ ID NO:

79.

39. 37. The polynucleotide of Claim 36, wherein the first CAR and the second CAR are encoded on a single vector.

40. 40. The polynucleotide of claim 39, wherein the vector is a viral vector, a lentiviral vector, a non-viral vector, or a transposon.

41. 41. The polynucleotide of claim 40, wherein the vector is a bicistronic lentiviral vector.

42. 1. A method for generating a population of genetically modified immune cells, comprising: a) introducing a composition comprising the polynucleotide sequence of claim 36 into a plurality of immune cells, thereby generating a population of genetically modified immune cells; b) culturing the population of genetically modified immune cells under conditions suitable for integration of the polynucleotide sequence; c) expanding and / or selecting at least one cell from the population of genetically modified immune cells that expresses the first CAR and the second CAR on the surface of the cell; and A method comprising:

43. 34. The composition of claim 33 for the treatment of cancer in a subject in need thereof.

44. 44. The composition of claim 43, wherein administration of a composition comprising modified immune cells comprising the first CAR and the second CAR increases an immune response against the target cells compared to administration of a composition comprising modified immune cells comprising only the first CAR.

45. 45. The composition of claim 44, wherein the increased immune response is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage therebetween, greater than a composition comprising an engineered immune cell comprising only the first CAR.

46. 44. The composition of claim 43, wherein the cancer is a solid tumor, a B-cell malignancy, a myeloid malignancy, a T-cell malignancy, acute lymphoblastic leukemia, acute lymphoblastic lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, chronic myeloid leukemia, T-lymphoblastic leukemia, T-lymphoblastic lymphoma, or anaplastic large cell leukemia.

47. 44. The composition of claim 43, wherein the cancer has low cell surface expression of the first antigen and / or low cell surface expression of the second antigen.

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