Application of CD2 / 5 / 7 knockout anti-CD2 / 5 / 7 chimeric antigen receptor T cell to T cell lymphoma and leukemia

Abstract

The invention relates to application of a CD2 / 5 / 7 knockout anti-CD2 / 5 / 7 chimeric antigen receptor T cell to T cell lymphoma and leukemia. The present invention includes compositions and methods for treating T cell lymphoma and leukemia. In certain aspects, the compositions and methods include CAR T cells that target CD2, CD5, or CD7 and modified cells in which CD2, CD5, or CD7 has been knocked out.

Description

[0001] This application is a continuation-in-part of patent application number 2019800922819, filed December 19, 2019, entitled "USE OF CD2 / 5 / 7 KNOCKOUT ANTI-CD2 / 5 / 7 CHIMERIC ANTIGEN RECEPTOR T CELLS AGAINST T CELL LYMPHOMAS AND LEUKEMIAS."

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 782,131, filed December 19, 2018, which is incorporated by reference in its entirety. BACKGROUND

[0004] T cell lymphomas and leukemias are aggressive neoplasms derived from T cell progenitors or differentiated T cells. Mature or peripheral T cell lymphomas account for 10-15% of all non-Hodgkin lymphomas, or approximately 7,000 to 10,000 cases per year in the United States. T cell lymphomas and leukemias have a poor prognosis and few available treatments. Chimeric antigen receptor T cell (CART) therapy has proven effective against B cell neoplasms, but successful extension of CAR T cells to T cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T cell fratricide.

[0005] There is a need for compositions and methods for treating T cell lymphomas and leukemias and for methods to eliminate CAR T cell fratricide. The present invention addresses this need. SUMMARY

[0006] As described herein, the present invention relates to compositions and methods utilizing CAR T cells targeting CD2, CD5, or CD7 and modified cells in which CD2, CD5, or CD7 has been knocked out.

[0007] In one aspect, the present invention includes a method of treating a cancer in a subject in need thereof. The method includes administering to the subject a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell in which an endogenous CD5 gene has been knocked out.

[0008] In another aspect, the present invention includes a method of treating a cancer in a subject in need thereof. The method includes administering to the subject a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain capable of binding CD2, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell in which an endogenous CD2 gene has been knocked out.

[0009] In yet another aspect, the application includes a method of treating cancer in a subject in need thereof. The method includes administering to the subject a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain capable of binding CD5, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell, wherein the endogenous CD5 gene has been knocked out.

[0010] In yet another aspect, the application includes a method of treating cancer in a subject in need thereof. The method includes administering to the subject a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain capable of binding CD5, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell, wherein the endogenous CD5 gene has been knocked out.

[0011] Another aspect of the application includes a nucleic acid comprising a CAR, wherein the CAR comprises an antigen binding domain capable of binding CD2, a transmembrane domain, and an intracellular domain.

[0012] Yet another aspect of the application includes a nucleic acid comprising a CAR, wherein the CAR comprises an antigen binding domain capable of binding CD5, a transmembrane domain, and an intracellular domain.

[0013] Still another aspect of the application includes a vector comprising any of the nucleic acids disclosed herein.

[0014] In another aspect, the application includes a cell comprising any of the nucleic acids disclosed herein or any of the vectors disclosed herein.

[0015] In yet another aspect, the application includes a composition comprising a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain targeting CD2, a transmembrane domain, and an intracellular domain; and a second modified cell, wherein the endogenous CD2 gene has been knocked out.

[0016] In yet another aspect, the application includes a composition comprising a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain targeting CD5, a transmembrane domain, and an intracellular domain; and a second modified cell, wherein the endogenous CD5 gene has been knocked out.

[0017] In yet another aspect, the application includes a composition comprising a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain targeting CD7, a transmembrane domain, and an intracellular domain; and a second modified cell, wherein the endogenous CD7 gene has been knocked out.

[0018] Another aspect of the application includes a composition comprising a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell, wherein an endogenous CD5 gene has been knocked out.

[0019] In various embodiments of the above aspects or any other aspect of the application described herein, the endogenous gene is knocked out using a CRISPR method. In certain embodiments, the CRISPR method is a CRISPR / Cas9 method. In certain embodiments, the CRISPR / Cas9 method utilizes an sgRNA comprising the nucleotide sequence of SEQ ID NO: 23. In certain embodiments, the CRISPR / Cas9 method utilizes an sgRNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22-24.

[0020] In certain embodiments, the antigen binding domain of the CAR is capable of binding an antigen selected from the group consisting of CD5, CD19, CD2, CD7, tumor specific antigen (TSA), tumor associated antigen (TAA), a glioma-associated antigen, carcinoembryonic antigen (CEA), b-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, muthsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin, telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBV A, HPV antigen E6, HPV antigen E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-catenin, CDK4, Mum-1, p 15, p16, 43-9F, 5T4, 791Tgp72, alphafetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0021] In certain embodiments, the antigen binding domain of the CAR comprises a complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36, 43-48, 53-58, 65-70, 83-88, and 95-100. In certain embodiments, the antigen binding domain of the CAR comprises a complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36, 43-48, 53-58, and 65-70. In certain embodiments, the antigen binding domain of the CAR comprises a complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-88 and 95-100.

[0022] In certain embodiments, the antigen binding domain of the CAR comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 41, 51, 63, 75, 81, and 93. In certain embodiments, the antigen binding domain of the CAR comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 42, 52, 64, 76, 82, and 94.

[0023] In certain embodiments, the antigen binding domain of the CAR comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 41, 51, and 63. In certain embodiments, the antigen binding domain of the CAR comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 42, 52, and 64.

[0024] In certain embodiments, the antigen binding domain of the CAR comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 75, 81, and 93. In certain embodiments, the antigen binding domain of the CAR comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 82, and 94.

[0025] In certain embodiments, the antigen binding domain of the CAR comprises a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79, 80, 91, and 92. In certain embodiments, the antigen binding domain of the CAR comprises a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, and 62. In certain embodiments, the antigen binding domain of the CAR comprises a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 74, 79, 80, 91, and 92.

[0026] In certain embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 37, 38, 49, 59, 60, 71, 72, 77, 78, 89, and 90. In certain embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 37, 38, 49, 59, and 60. In certain embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71, 72, 77, 78, 89, and 90.

[0027] In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 13. In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7. In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 to 13.

[0028] In certain embodiments, the CAR further comprises a suicide gene. In certain embodiments, the suicide gene is iCaspase9.

[0029] In certain embodiments, the first and / or second modified cell is a T cell.

[0030] In certain embodiments, the cancer comprises a T cell lymphoma or a T cell leukemia. In certain embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0031] In certain embodiments, the composition comprises a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF DRAWINGS

[0032] The following detailed description of specific embodiments of the application will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the application, exemplary embodiments are shown in the drawings. It should be understood, however, that the precise arrangements and instrumentalities shown in the drawings are not intended to limit the application to the precise arrangements and instrumentalities shown.

[0033] Figure 1 is a schematic illustration of the current problem exemplifying CART therapy for T cell tumors.

[0034] Figure 2 is a schematic illustration of the development of an example innovative strategy, wherein: i. the tumor target is removed from normal T cells using gene editing and thereby avoiding fratricide in the manufacturing process; ii. a second T cell product containing normal T cells, wherein the T cell target is knocked out (KO), is co-infused with the anti-T cell tumor CART to provide T cell immunity that is not affected by the CART killing.

[0035] Figure 3 is a schematic of an example new approach to target T cell lymphomas without causing T cell toxicity. A two-pronged immunotherapy is used, which includes anti-T-NHL (or T-ALL) CART (one of CART2 / 5 / 7, e.g., CART2) and CD2 / 5 / 7 knockout normal T cells. The CART will destroy tumor cells, but will also kill normal T cells. Infusion of CD2 / 5 / 7 (or other tumor target) KO normal T cells will provide CART resistant T cell immunity until the CART cells are exhausted.

[0036] Figure 4 Example anti-CD2 and anti-CD5 CAR constructs used herein are shown. All constructs have a lentiviral pTRPE4-1BB CD3 zeta backbone.

[0037] Figure 5 CART transduction efficiency in T cells is exemplified. Six different CAR5 constructs were generated using single chain variable fragments (scFv) with high (#17), medium (#34), and low (#9) affinity. T cells were activated with anti-CD3 / CD28 beads (Dynabeads), and 24 hours later lentiviral vector was added at an MOI of 3. Dynabeads were removed at day 6. When average volume was below 350 fl, CART cells were frozen. CAR expression was tested at day 6 (goat anti-mouse Fab antibody).

[0038] Figure 6 CD5 (or CD2, or CD7) KO manufacturing process and CRISPR-Cas9 KO efficiency is exemplified.

[0039] Figure 7 Expansion curves for several CART groups are exemplified. Without CD2 KO, CART2 cells do not expand. With KO, CART2 and CART5 reach about 5-8 fold population doubling.

[0040] Figure 8 The following finding is exemplified: with CRISPR-Cas9 KO of CD5, CD5 mean fluorescence intensity (MFI) in CART5 is 10-fold lower compared to control T cells, while another pan T cell marker (such as CD2) is unchanged.

[0041] Figure 9 CART2 and CART5 expansion curves are exemplified. T cell concentration was measured using a Coulter Counter.

[0042] Figure 10Results from experiments are illustrated where six different CAR2 constructs were challenged in vitro by co-culturing them with luciferase+ Jurkat cells (a T cell leukemia cell line). Total killing was measured as relative reduction in luminescence at 24 hours. Only C3029, C3030 and C3043 showed anti-tumor effects.

[0043] Figure 11 Results from experiments are illustrated where six different CAR5 constructs were challenged in vitro by co-culturing them with luciferase+ Jurkat cells (a T cell leukemia cell line). Total killing was measured as relative reduction in luminescence at 24 hours. All CAR5 constructs showed similar anti-tumor effects.

[0044] Figure 12 In vivo efficacy of CART2 and CART5 is illustrated. NSG mice were engrafted with luciferase+ Jurkat cells and mice were randomized on day 7 to receive control T cells or CART2 or CART5 (1x10 6 ) weekly using IVIS Xenogen Spectrum and analyzed using Livinglmage software. CART2 C3043 and CART5 C3054 were the most effective.

[0045] Figure 13 Results from experiments are illustrated where Jurkat cells were transduced with different CAR5 constructs (targeting epitope and affinity shown on left) and GFP-NFAT reporter and then co-cultured with CD5+ tumor cells (or control) for 24 hours. The lead CART5 (C3054) showed increased NFAT activation.

[0046] Figure 14 Results from experiments are illustrated where Jurkat cells were transduced with different CAR2 constructs and GFP-NFAT reporter and then co-cultured with CD2+ tumor cells (or control) for 24 hours. The lead CART2 (C3043) showed increased NFAT activation.

[0047] Figure 15 CART2 and CART5 activity against cutaneous T cell lymphoma is illustrated. Results of a 24 hour killing assay are shown. CART2 cells were active against primary Sezary cells (leukemic cutaneous T cell lymphoma) and HH Sezary cell line. CART5 was also active against HH cells.

[0048] Figure 16The following finding was exemplified: CART2 and CART5 can recognize and kill normal T cells (autologous top and allogeneic bottom).

[0049] Figure 17 The following finding was exemplified: Removal of CAR targets protects normal T cells from CART killing. CD5 KO but not WT normal T cells are resistant to CART5 killing. Normal resting T cells are recognized and killed by CART2 (top) and CART5 (bottom). Efficient KO of CD2 or CD5 from normal T cells using CRISPR-Cas9 results in resistance to CART2 or CART5 killing, respectively.

[0050] Figure 18 The following finding was exemplified: CMV-specific T cells exist in CD2 KO and CD5 KO normal T cell products. CD2 and CD5 KO normal T cells maintain the ability to recognize CMV peptides and produce cytokines. (HLA-A-02:01-CMV PP65 NLVPMVATV dextramer (SEQ ID NO: 101); ICS after 4 hours of exposure to CETF peptides. After secondary culture with CMV-peptide pulsed APCs).

[0051] Figure 19 The development of bispecific CAR T cells was exemplified. Two lentivirus constructs were generated that include CAR5 (C3054) and CAR2 (C3043) linked by a P2A sequence. Gene expression is driven by an EF1 alpha promoter. The CAR5 construct has a 4-1BB costimulatory domain and a CD3 zeta signaling domain.

[0052] Figures 20A-20B BisKO CART cells were exemplified. Efficient knockout of CD2 and CD5 in normal T cells was shown by flow cytometry.

[0053] Figure 21 The following finding was exemplified: CD5 KO CART5 is more potent than CD5+ CART5 in vivo. CD5 KO increases the anti-tumor efficacy of CART5. In a Jurkat T-ALL xenograft model using NSG mice, CD5 KO CART5 (2x10 6 cells / mouse) resulted in complete long-term complete responses and longer survival compared to WT CART5.

[0054] Figure 22The following finding was exemplified: CD5 KO CART 19 is more effective than CD5+ CART 19 in vivo. CD5 KO increases the anti-tumor efficacy of CART 19. CD5 KO CART 19 has significantly higher tumor control compared to WT CART 19 in the NALM6 B-ALL xenograft model.

[0055] Figures 23A-23B The following finding was exemplified: CART5 and CART2 can target 20% of AML. Figure 23A CD2 expression in AML was exemplified. Figure 23B Results from a 24 hour killing assay were exemplified. CART2 cells were co-cultured with CD2+ AML cells and showed significant killing at 24 hours.

[0056] Figure 24 The following finding was exemplified: CART5 can target 100% of CLL and MCL. Results from cytotoxicity assays showing that CART5 cells can recognize and kill CD5+ MCL cell lines (Jeko-1 and Mino).

[0057] Figure 25 Development of sgRNAs to knock out CD7 in T cells (top) and generation of six CAR constructs against CD7 were exemplified.

[0058] Figure 26 A Casp9-CAR5 lentivirus construct used herein was exemplified. Two lentivirus constructs were generated, including CAR5 (C3054), a P2A sequence, and then either iCaspase9 suicide gene (iC9) or iC9-P2A-C3054. Gene expression is driven by an EF1 alpha promoter. The CAR5 construct has a 4-IBB costimulatory domain and a CD3 zeta signaling domain. DETAILED DESCRIPTION

[0059] Definitions

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present application, the preferred materials and methods are described herein. In describing and claiming the present application, the following terminology will be used.

[0061] It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0062] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0063] "About", as used herein, when referring to a measurable value such as an amount, a time period, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1% and yet more preferably ±0.1% from the specified value, as long as such variations are appropriate for the method being described.

[0064] "Activated", as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cell" and the like refer to a T cell that undergoes cell division.

[0065] The term "antibody", as used herein, refers to an immunoglobulin molecule that specifically binds an antigen. Antibodies can be intact immunoglobulins derived from natural sources, or derived from recombinant sources. Antibodies can be immunologically reactive portions of the intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present application can exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0066] The term "antibody fragment" refers to a portion of an intact antibody, and refers to the antigenic determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies formed from antibody fragments, scFv antibodies, and multi-specific antibodies.

[0067] "Antibody heavy chain", as used herein, refers to the larger of the two types of polypeptide chains found in all antibody molecules in their naturally occurring conformation.

[0068] "Antibody light chain," as used herein, refers to the smaller chain of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation, kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0069] The term "synthetic antibody" as used herein means an antibody produced using recombinant DNA technology, such as for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody that has been produced from the synthesis of a DNA molecule, the DNA molecule encoding the antibody and the DNA molecule expressing the antibody protein or specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology available and well known in the art.

[0070] The term "antigen" or "Ag" as used herein is defined as a molecule that provokes an immune response, which can involve antibody production, or activation of specific immune-competent cells, or both. The skilled artisan will appreciate that any macromolecule - virtually including all proteins or peptides - can serve as an antigen. Furthermore, an antigen can be derived from recombinant or genomic DNA. The skilled artisan will appreciate that any DNA - which includes a nucleotide sequence encoding a protein that elicits an immune response, or a portion of a nucleotide sequence, thus encoding an antigen as the term is used herein. Furthermore, the skilled artisan will appreciate that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It will be readily apparent that the present application includes, but is not limited to, the use of portions of nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to elicit the desired immune response. Furthermore, the skilled artisan will appreciate that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be produced, synthesized, or can be derived from a biological sample. Such a biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0071] The term "autologous" as used herein means with respect to any material derived from the same individual to which it is subsequently reintroduced.

[0072] "Heterologous" refers to any material derived from a different animal of the same species.

[0073] "Heterologous" refers to any material derived from a different animal of the same species.

[0074] The term "chimeric antigen receptor" or "CAR", as used herein, refers to an artificial T cell receptor engineered to be expressed on an immune effector cell and specifically binds to an antigen. CARs can be used as a therapy for adoptive cell transfer. T cells are removed from a patient and modified so that they express a receptor specific for a particular form of an antigen. In some embodiments, the CAR is specific for a selected target, e.g., a B cell surface receptor. CARs can also comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region. In some aspects, the CAR comprises an extracellular domain comprising an anti-B cell binding domain fused to a CD3-zeta transmembrane domain and intracellular domain.

[0075] The term "cleave" refers to the breaking of a covalent bond, such as in the backbone of a nucleic acid molecule or the hydrolysis of a peptide bond. Cleavage can be initiated by a variety of methods, including but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Single strand cleavage and double strand cleavage are both possible. Double strand cleavage can occur as a result of two distinct single strand cleavage events. DNA cleavage can result in the production of either blunt or staggered ends. In certain embodiments, fusion polypeptides can be used to target cleaved double stranded DNA.

[0076] As used herein, the term "conservative sequence modifications" is intended to indicate alterations of the amino acid sequence that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibody of the application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CDR region of an antibody can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for its ability to bind antigen using the functional assays described herein.

[0077] The term "costimulatory ligand" as used herein includes a molecule on an antigen presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) that specifically binds a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response in addition to the primary signal provided by, for example, the binding of a TCR / CD3 complex to a peptide-loaded MHC molecule, including but not limited to proliferation, activation, differentiation, etc. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll ligand receptor, and a ligand that specifically binds to B7-H3. Costimulatory ligands also encompass, among others, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0078] A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0079] As used herein, a "costimulatory signal" refers to a signal that, in conjunction with a primary signal, such as TCR / CD3 engagement, results in T cell proliferation and / or up- or down-regulation of key molecules.

[0080] A "disease" is a state of health of an animal in which the animal is unable to maintain homeostasis, and in which the health of the animal continues to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the state of health of the animal is less favorable than it would be if it were not in the disorder. Left untreated, a disorder does not necessarily result in a further decrease in the state of health of the animal.

[0081] As used herein, the term "down-regulation" refers to a decrease or elimination of gene expression of one or more genes.

[0082] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition, as described herein, effective in achieving a particular biological result or providing a therapeutic or prophylactic benefit. Such results can include, but are not limited to, anti-tumor activity as determined by any suitable means in the art.

[0083] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having the same or approximately the same

[0084] "Endogenous," as used herein, refers to any substance that is from or produced within an organism, cell, tissue, or system.

[0085] The term "exogenous," as used herein, refers to any substance introduced into or produced outside of an organism, cell, tissue, or system.

[0086] The term "expand," as used herein, refers to an increase in number, such as an increase in the number of T cells. In one embodiment, the ex vivo expanded T cells increase in number relative to the number initially present in culture. In another embodiment, the ex vivo expanded T cells increase in number relative to other cell types in culture. The term "ex vivo," as used herein, refers to cells that have been removed from a living organism (e.g., a human) and propagated outside of the organism (e.g., in a petri dish, test tube, or bioreactor).

[0087] The term "express," as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0088] An “expression vector” refers to a vector that includes a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all of those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) incorporating the recombinant polynucleotide.

[0089] “Homologous,” as used herein, refers to subunit sequence identity between two polymeric molecules (e.g., between two nucleic acid molecules (such as between two DNA molecules or between two RNA molecules), or between two polypeptide molecules). When a position in both of two molecules is occupied by the same monomer subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half of the positions in two sequences are matched or homologous, then the two sequences have a homology of 50%; if 90% of the positions are matched or homologous, the sequences have a homology of 90%.

[0090] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 :522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0091] "Fully human" refers to an immunoglobulin, such as an antibody, in which the entire molecule is of human origin or consists of the same amino acid sequence as a human form of the antibody.

[0092] As used herein, "identity" refers to the subunit sequence identity between two polymeric molecules, particularly between two amino acid molecules (such as between two polypeptide molecules). When two amino acid sequences are the same at a given position; for example, if the position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or degree of identity between two amino acid sequences at the same position in an alignment is typically expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions (e.g., five positions in a polymer of ten amino acids) are occupied by the same amino acid in two sequences, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10) are occupied by the same amino acids, then the two amino acid sequences are 90% identical.

[0093] The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody found in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of most subjects. It is the most efficient immunoglobulin in agglutination reactions, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function, but can serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing the release of mediators from mast cells and basophils upon exposure to an allergen.

[0094] The term "immune response" as used herein is defined as a cellular response to an antigen that occurs when lymphocytes recognize an antigen molecule as foreign and induce antibody formation and / or activate lymphocytes to remove the antigen.

[0095] When referring to an "immunologically effective amount" or "therapeutic amount," the precise amount of the composition of the application to be administered can be determined by a physician or researcher taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in condition of the patient (subject).

[0096] As used herein, "instructional material" includes publications, records, graphs, or any other medium or means that conveys information to the user of the compositions and methods of the application. Instructional material can be included with the kit of the application, for example, as a package insert, in the container housing the nucleic acids, peptides, and / or compositions of the application, or as a package label associated with the container. Alternatively, the instructional material can be separate from the container(s), with the understanding that the material and the composition are intended to be used in conjunction with one another.

[0097] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or, for example, in a non-native environment such as a host cell.

[0098] The term "knockdown" as used herein refers to a reduction in gene expression of one or more genes.

[0099] The term "knockout" as used herein refers to the elimination of gene expression of one or more genes.

[0100] As used herein, "lentivirus" refers to the genus of the Retroviridae family. Among the retroviruses, lentiviruses are the only ones capable of infecting non-dividing cells; they can deliver significant amounts of genetic information into the DNA of the host cell, so they are one of the most efficient methods for gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses. Vectors derived from lentiviruses provide a tool to accomplish significant levels of gene transfer in vivo.

[0101] As used herein, the term "limited toxicity" means that the peptides, polynucleotides, cells and / or antibodies of the present application exhibit in vitro or in vivo a lack of substantially negative biological effects, anti-tumor effects or substantially negative physiological symptoms on healthy cells, non-tumor cells, non-diseased cells, non-target cells or populations of such cells.

[0102] As used herein, the term "modified" means an altered state or structure of a molecule or cell of the present application. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0103] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of a response in a subject as compared to the level of the response in the subject in the absence of treatment or a compound, and / or as compared to the level of the response in an otherwise identical subject that is not treated. The term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in the subject, preferably a human.

[0104] In the context of the present application, the following abbreviations are used for the commonly occurring nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0105] Unless otherwise indicated, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences which are degenerate versions of each other and which encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or an RNA can also include an intron(s) to the extent that the nucleotide sequence encoding the protein can in some version comprise the intron(s).

[0106] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is located in the same reading frame as the second nucleic acid sequence to permit translation of a fusion protein. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where present, functionally interrelated.

[0107] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate an abnormal level of tumor antigen expression in cells from a solid tumor within a particular tissue or organ of a patient relative to the level of expression from normal cells of the tissue or organ. A patient having a solid tumor or hematological malignancy characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0108] "Parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0109] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide are used interchangeably herein. One of skill in the art has general knowledge that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotides as used herein include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means, i.e., from a recombinant library or a cell genome, using ordinary cloning techniques and PCR TM and the like to clone nucleic acid sequences, and synthetic means.

[0110] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a sequence of a protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids that are linked to one another by peptide bonds. As used herein, the term refers to short chains, which are also sometimes referred to in the art as peptides, oligopeptides, and oligomers; and to longer chains, which are also sometimes referred to in the art as proteins, which have a variety of types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0111] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthesis machinery of the cell or an introduced synthesis machinery, which is required to initiate specific transcription of a polynucleotide sequence.

[0112] "Signaling pathway" refers to the biochemical relationship between various signaling molecules that act in the process of transmitting a signal from one part of a cell to another. The phrase "cell surface receptor" includes molecules and molecular complexes that are capable of receiving a signal across the plasma membrane of a cell and transmitting the signal.

[0113] As used herein, the term "specifically binds" with respect to an antibody means an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species can also bind antigens from one or more species. However, such cross-species reactivity does not by itself change the class of specificity of the antibody. In another example, an antibody that specifically binds an antigen can also bind different allelic forms of the antigen. However, such cross-reactivity does not by itself change the class of specificity of the antibody. In some examples, the term "specifically binds" or "binds specifically" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody typically recognizes and binds a specific protein structure rather than a protein in general. If an antibody is specific for an epitope "A", the presence of a molecule containing epitope A (or free, unlabelled A) in a reaction containing labelled "A" and the antibody will decrease the amount of labelled A bound to the antibody.

[0114] By the term "stimulation" is meant the induction of a primary response by binding of a stimulatory molecule (e.g., TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as but not limited to signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as down-regulation of TGF-β and / or reorganization of cytoskeletal structures, among others.

[0115] "Stimulatory molecule", as the term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

[0116] As used herein, "stimulatory ligand" means a ligand that, when present on an antigen presenting cell (e.g., aAPC, dendritic cell, B-cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and encompass, among others, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0117] The term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0118] As used herein, a "substantially purified" cell is a cell that is substantially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cells that normally associate with it in its naturally occurring state. In some examples, a population of substantially purified cells refers to a homogeneous population of cells. In other examples, the term simply refers to cells that have been separated from the cells that normally associate with them in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0119] A "target site" or "target sequence" refers to a genomic nucleic acid sequence defining a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0120] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins that are involved in the activation of T cells to antigen presentation. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. The TCR consists of a heterodimer of alpha (a) and beta (β) chains, but in some cells the TCR consists of gamma and delta (γ / δ) chains. The TCR can exist in a and β and γ / δ forms, which are structurally similar but have different anatomical locations and functions. Each chain consists of two extracellular domains, a variable domain, and a constant domain. In some embodiments, the TCR can be modified on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0121] As used herein, the term "therapeutic" means treatment and / or prophylaxis. A therapeutic effect is obtained by inhibiting, alleviating or eliminating a disease state.

[0122] The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the original subject cell and its progeny.

[0123] The term "treatment" of a disease as used herein means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0124] The phrase "under the transcriptional control" or "operably linked" as used herein means that a promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription of a RNA polymerase and the expression of the polynucleotide.

[0125] A "vector" is a composition of matter comprising an isolated nucleic acid, and which is useful in delivering the isolated nucleic acid into the interior of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionizable or amphiphilic compound, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

[0126] RANGES: Throughout this disclosure, aspects of the application can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and does not

[0127] Description

[0128] The present disclosure describes three chimeric antigen receptors (CARs) that target T cell neoplasms and methods to protect healthy T cells from fratricide. T cell lymphomas and leukemias are aggressive neoplasms that arise from T cell progenitors or differentiated T cells. Mature or peripheral T cell lymphomas account for 10-15% of all non-Hodgkin lymphomas, or approximately 7,000 to 10,000 cases per year in the United States. T cell lymphomas and leukemias have a poor prognosis and few available treatments. CAR T therapy has proven efficacious against B-cell neoplasms, but until now, there have been problems extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies because most target antigens are shared between normal and malignant cells, leading to CAR T cell fratricide. Here, CRISPR-Cas editing is used to remove target antigens from healthy T cells, protecting them from CAR T cell therapy and eliminating the potential for fatal immune suppression resulting from the elimination of the T-cell compartment.

[0129] In certain embodiments, the CAR targets the T cell antigens CD2, CD5, and CD7. In certain embodiments, CRISPR-Cas knockout of the CD2, CD5, or CD7 target in healthy T cells prevents killing of healthy T cells during manufacturing and subsequent CAR T therapy.

[0130] Methods of Treatment

[0131] The present application includes methods of treating T cell lymphoma or T cell leukemia in a subject in need thereof. In another aspect, the present application includes methods of preventing CAR T cell fratricide in a subject in need thereof.

[0132] In certain embodiments, the method comprises administering to the subject a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain targeting CD2, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell in which an endogenous CD2 gene has been knocked out.

[0133] In certain embodiments, the method comprises administering to the subject a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain targeting CD5, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell in which an endogenous CD5 gene has been knocked out.

[0134] In certain embodiments, the method comprises administering to the subject a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain targeting CD7, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell in which an endogenous CD7 gene has been knocked out.

[0135] In certain embodiments, the method comprises administering to the subject a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell in which an endogenous CD5 gene has been knocked out.

[0136] In certain embodiments, the method comprises administering to the subject a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and wherein an endogenous CD5 gene in the cell has been knocked out.

[0137] In various embodiments of the methods disclosed herein, any of the CARs disclosed herein can be administered to the subject. The CARs are specific for any tumor associated antigen (TAA) or tumor specific antigen (TSA) known to one of ordinary skill in the art.

[0138] In certain embodiments, the CAR comprises a complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36, 43-48, 53-58, 65-70, 83-88, and 95-100. In certain embodiments, the CAR comprises an antigen binding domain comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 41, 51, 63, 75, 81, and 93 and / or a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 42, 52, 64, 76, 82, and 94. In certain embodiments, the CAR comprises a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79, 80, 91, and 92.

[0139] In certain embodiments, the subject is administered a CAR, wherein the CAR comprises a nucleic acid sequence encoded by any one of SEQ ID NOs: 1-13. In certain embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 37, 38, 49, 59, 60, 71, 72, 77, 78, 89, and 90.

[0140] In certain embodiments, the first and / or second modified cell is a T cell. In certain embodiments, the cancer comprises a T cell lymphoma or a T cell leukemia. Types of cancer that can be treated with the compositions and methods of the application include, but are not limited to, non-Hodgkin’s lymphoma and subtypes thereof, including peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL, ALK-), natural killer / T-cell lymphoma (NKTCL), adult T-cell leukemia / lymphoma (ATLL), ALCL ALK+, enteropathy-type T-cell, hepatosplenic T-cell, subcutaneous panniculitis-like, and unclassified PTCL.

[0141] In certain embodiments, the endogenous genes (e.g., CD2, CD5, and CD7) are knocked out using a CRISPR / Cas9 method. In certain embodiments, the CRISPR / Cas9 method utilizes sgRNAs targeting CD2, CD5, and / or CD7. In certain embodiments, the sgRNAs comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22-24.

[0142] In certain embodiments, the CAR of the application further comprises a suicide gene. One non-limiting example of a suicide gene is the inducible Caspase 9 gene (iCaspase9, iCasp9, or iC9). The iCaspase9 suicide gene system is based on a fusion of human caspase 9 with a modified human FK-binding protein, allowing conditional dimerization using a small molecule drug (e.g., API 903). When exposed to the synthetic dimerizing drug, iCaspase9 becomes activated and leads to rapid apoptosis of cells (e.g., CAR T cells) expressing the construct (Zhou et al. (2015) Methods Mol Biol. 1317:87-105). Another example of a suicide gene is the HSV-tk gene (Bordingnon et al. (1995) Human Gene Therapy, vol. 6, no. 6., pp 813-819). The HSV-tk gene can be co-expressed in CAR T cells, and upon expression it converts the nontoxic prodrug GCV to GCV-triphosphate, leading to cell death by stopping DNA replication.

[0143] Compositions

[0144] One aspect of the application includes a composition comprising a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain targeting CD2, a transmembrane domain, and an intracellular domain; and a second modified cell, wherein the endogenous CD2 gene has been knocked out.

[0145] Another aspect of the application includes a composition comprising a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain targeting CD5, a transmembrane domain, and an intracellular domain; and a second modified cell, wherein the endogenous CD5 gene has been knocked out.

[0146] Yet another aspect of the application includes a composition comprising a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain targeting CD7, a transmembrane domain, and an intracellular domain; and a second modified cell, wherein the endogenous CD7 gene has been knocked out.

[0147] In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 13. In certain embodiments, the endogenous gene has been knocked out using a CRISPR / Cas system. In certain embodiments, the CRISPR / Cas9 system comprises a gRNA comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22 to 24.

[0148] The application further includes a composition of the application and a pharmaceutically acceptable carrier.

[0149] Chimeric Antigen Receptors (CARs)

[0150] The present application provides a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the present application includes a CAR comprising an antigen binding domain capable of binding CD2, a transmembrane domain, and an intracellular domain.

[0151] Antigen Binding Domain

[0152] In one embodiment, the CAR of the present application comprises a target-specific binding element, also referred to as an antigen binding domain. The choice of antigen binding domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen binding domain can be selected to recognize a ligand that functions as a cell surface marker on a target cell associated with a particular disease state, such as a T cell lymphoma or leukemia.

[0153] In one embodiment, the CAR of the present application can be engineered to target a tumor antigen. The antigens discussed herein are included by way of example only. The list is not intended to be exclusive and further examples will be readily apparent to those skilled in the art. A tumor antigen is a protein produced by a tumor cell that can elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen binding domain of the present application will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin- reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0154] The tumor antigens referred to in the present application can also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not occur on other cells of the body. TAAs are antigens associated with tumors and, in contrast, are expressed on normal cells as well under conditions in which the immune system is unable to induce a state of immune tolerance to the antigen. Antigen expression on tumors can occur under conditions in which the immune system is able to respond to the antigen. TAAs can be antigens expressed on normal cells during embryonic development when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0155] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-l / MelanA (MART-1), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigens EBVA and human papilloma virus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCA, CA 195, CA 242, CA-50, CAM43, CD68 PI, CO-029, FGF-5, G250, Ga733 EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 Mac-2 binding protein, cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0156] Depending on the desired antigen to be targeted, the CAR of the application can be engineered to include an appropriate antigen binding domain specific for the desired antigen target. For example, if CD2 is the desired antigen to be targeted, an antibody against CD2 can be used as the antigen binding domain for incorporation into the CAR of the application.

[0157] In certain embodiments, the antigen binding domain of the CAR targets CD2. In certain embodiments, the antigen binding domain of the CAR targets CD5. In certain embodiments, the antigen binding domain of the CAR targets CD7.

[0158] In some embodiments, the antigen binding domain in the CAR of the application is an anti-CD2 scFV. In some embodiments, the antigen binding domain in the CAR of the application is an anti-CD5 scFV. In some embodiments, the antigen binding domain in the CAR of the application is an anti-CD7 scFV. In some embodiments, the antigen binding domain is an anti-CD2 antibody. In some embodiments, the antigen binding domain is an anti-CD5 antibody. In some embodiments, the antigen binding domain is an anti-CD7 antibody.

[0159] In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs).

[0160] In certain embodiments, the application includes a CAR comprising an antigen binding domain capable of binding to CD2, wherein the antigen binding domain comprises a complementarity determining region (CDR) comprising the amino acid sequence of any one of SEQ ID NOs: 31, 32, 33, 34, 35, 36, 43, 44, 45, 46, 47, 48, 53, 54, 55, 56, 57, 58, 65, 66, 67, 68, 69, or 70.

[0161] In certain embodiments, the CAR comprises an antigen binding domain capable of binding to CD2, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 31, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 32, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 33, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 36.

[0162] In certain embodiments, the CAR comprises an antigen binding domain that is capable of binding CD2, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 43, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 44, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 45, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 46, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 48.

[0163] In certain embodiments, the CAR comprises an antigen binding domain that is capable of binding CD2, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 65, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 66, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 68, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 69, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 70.

[0164] In certain embodiments, the CAR comprises an antigen binding domain that is capable of binding CD2, wherein the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52. In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64.

[0165] In certain embodiments, the CAR comprises an antigen binding domain that is capable of binding CD2, wherein the antigen binding domain is a scFv, the scFv comprising the amino acid sequence set forth in any one of SEQ ID NOs: 27, 28, 39, 40, 50, 61, or 62.

[0166] In certain embodiments, the application includes a CAR comprising an antigen binding domain capable of binding CD5, wherein the antigen binding domain comprises a complementarity determining region (CDR) comprising the amino acid sequence of any one of SEQ ID NOs: 83, 84, 85, 86, 87, 88, 95, 96, 97, 98, 99, or 100.

[0167] In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD5, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 83, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 84, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 85, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 86, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 88.

[0168] In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD5, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 95, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 96, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 97, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 98, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 99, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 100.

[0169] In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD5, wherein the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 82. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 93 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 94.

[0170] In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD5, wherein the antigen binding domain is an scFv comprising the amino acid sequence recited in any one of SEQ ID NOs: 73, 74, 79, 80, 91, or 92.

[0171] Permitted variations of the antigen binding domain sequence will be known to those skilled in the art. For example, in some embodiments, the antigen binding domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 73, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0172] Transmembrane Domain

[0173] With respect to the transmembrane domain, the CAR can be designed to comprise a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0174] The transmembrane domain can be of natural or synthetic origin. When of natural origin, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region of particular use in the present application can be derived from the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., at least comprising a transmembrane region (one or more) of the above). Alternatively, the transmembrane domain can be synthetic, in which case it will comprise a preponderance of hydrophobic residues such as leucine and valine. Preferably, a triad of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, can form a junction between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine doublets provide particularly suitable linkers.

[0175] In one embodiment, the transmembrane domain in the CAR of the application is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 14. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 15. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 15.

[0176] In some cases, the transmembrane domain of the CAR of the application comprises a CD8a hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 16. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 17. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 17.

[0177] A spacer domain can be incorporated between the antigen binding domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally means any oligo- or polypeptide that serves to link the transmembrane domain to an extracellular or cytoplasmic domain in the polypeptide chain. The spacer domain can comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0178] Intracellular Domain

[0179] The intracellular domain or additional cytoplasmic domain of the CAR of the application is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. For example, an effector function of a T cell can be cytolytic activity or helper activity including cytokine secretion. Thus, the term "intracellular domain" refers to that portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. While generally the entire intracellular domain can be employed, in many instances it is not necessary to use the entire chain. In terms of using a truncated portion of the intracellular domain, such a truncated portion can be used in place of the entire chain so long as it transduces the effector function signal. Thus, the term intracellular domain is intended to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.

[0180] Preferred examples of intracellular domains for use in the CAR of the application include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that synergize to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capability.

[0181] It is known that the signal generated by the TCR alone is insufficient to fully activate T cells and that a secondary or costimulatory signal is also required. Thus, T cell activation can be considered to be mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).

[0182] Primary cytoplasmic signaling sequences modulate primary activation of the TCR complex either in a stimulatory manner or in an inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner can comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0183] Examples of ITAMs containing primary cytoplasmic signaling sequences that have particular use in the present application include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Particularly preferably, the cytoplasmic signaling molecule in the CAR of the application comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0184] In preferred embodiments, the intracellular domain of the CAR can be designed to comprise a CD3-zeta signaling domain by itself, or can be combined with any other desired intracellular domain(s) useful in the context of the CAR of the application. For example, the intracellular domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. Costimulatory signaling region refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. Thus, while the present application primarily uses 4-1BB as an example of a costimulatory signaling element, other costimulatory elements are within the scope of the present application.

[0185] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the application can be linked to each other in random or in a defined order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0186] In one embodiment, the intracellular domain in the CAR of the application is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0187] In one embodiment, the intracellular domain in the CAR of the application is designed to comprise the signaling domain of 4-IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO: 18 and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 19.

[0188] In one embodiment, the intracellular domain in the CAR of the application is designed to comprise the signaling domain of 4-IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-IBB comprises the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 20 and the signaling domain of CD3-zeta comprises the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 21.

[0189] In one embodiment, the intracellular domain in the CAR of the application is designed to comprise the signaling domain of 4-IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 20 and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0190] In one embodiment, the anti-CD2 CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 26, 37, 38, 49, 59, or 60. In one embodiment, the anti-CD2 CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7. In one embodiment, the anti-CD5 CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 71, 72, 77, 78, 89, or 90. In one embodiment, the anti-CD5 CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 to 13.

[0191] Permitted variations of the CAR sequences will be known to those of skill in the art. For example, in some embodiments, the CAR comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NO: 25, 26, 37, 38, 49, 59, 60, 71, 72, 77, 78, 89, or 90. In some embodiments, the CAR is encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0192] The present application should be interpreted to include any of the following: a CAR, a nucleic acid encoding a CAR, a vector comprising a nucleic acid encoding a CAR, a cell comprising a CAR, a cell comprising a nucleic acid encoding a CAR, and a cell comprising a vector comprising a nucleic acid encoding a CAR.

[0193] CD2-MEDI507 H2L-3028 CAR (SEQ ID NO: 1) CD2-OKT11 H2L-3029 CAR (SEQ ID NO: 2)

[0194] ggatccCAAGTCCAACTGGTGCAATCAGGCGCAGAAGTCCAACGACCGGGGGCCAGTGTTAAAGTGTCTTGTAAAGCCTCCGGGTACATTTTTACTGAGTACTATATGTACTGGGTCAGACAGGCCCCAGGGCAAGGTTTGGAACTTGTCGGACGCATAGATCCCGAAGACGGTTCTATAGATTACGTTGAGAAGTTCAAAAAGAAAGTCACACTTACTGCGGACACATCTAGTAGCACCGCATATATGGAACTGAGCAGTCTCACCTCAGACGACACCGCAGTGTACTATTGCGCTCGCGGAAAGTTTAACTATAGGTTCGCGTACTGGGGACAGGGGACACTGGTGACTGTTAGCAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGACGTTGTGATGACGCAAAGTCCCCCGTCACTCCTTGTTACTCTCGGCCAGCCAGCGTCTATCTCTTGCCGGTCAAGCCAGAGCTTGCTCCACTCTAGTGGTAACACGTATTTGAACTGGTTGCTGCAAAGGCCTGGACAATCTCCTCAGCCCCTGATCTATTTGGTTAGCAAACTGGAAAGTGGTGTTCCAGACAGATTTTCAGGGTCTGGATCAGGCACTGATTTCACTCTGAAGATCTCCGGGGTAGAGGCCGAGGACGTGGGAGTCTATTACTGCATGCAGTTTACTCACTATCCTTATACCTTTGGTCAAGGGACGAAACTGGAGATCAAAtccgga

[0195] CD2-OKT11 L2H-3030 CAR (SEQ ID NO: 3) CD2-T11-2-H2L-3031 CAR (SEQ ID NO: 4) CD2-TS2-18.1.1-H2L-3032

[0196] ggatccCAAGTTCAGCTTCAGCAACCAGGTGCTGAATTGGTCCGCCCTGGAACTAGCGTTAAACTGTCTTGTAAGGCATCCGGTTATACGTTTACAAGTTATTGGATGCACTGGATTAAGCAAAGGCCCGAACAAGGCCTTGAATGGATTGGGAGAATTGATCCCTACGATAGCGAGACACACTACAATGAAAAATTTAAAGATAAGGCCATCCTCAGCGTAGATAAGAGCAGTTCTACCGCATACATACAGCTCTCAAGCCTGACGTCAGATGACTCAGCCGTTTATTATTGCTCAAGGCGGGACGCTAAATACGACGGCTATGCGCTTGACTACTGGGGACAAGGCACCACTTTGACAGTCTCCAGTggtggcggagggagcggcggtggaggaagcggaggcggaggttccGATATAGTTATGACGCAAGCAGCACCCTCTGTACCTGTGACACCGGGTGAATCCGTTAGTATCTCATGCCGCTCTTCTAAAACCCTCTTGCATTCTAACGGCAATACATATTTGTATTGGTTCCTTCAACGACCAGGACAATCACCGCAAGTGCTTATTTATAGGATGTCTAACTTGGCTAGTGGGGTGCCAAATAGGTTCAGTGGGTCTGGATCTGAGACAACTTTCACGTTGAGAATAAGTAGGGTGGAAGCTGAAGACGTCGGTATATACTACTGTATGCAGCATTTGGAGTACCCTTACACTTTCGGGGGAGGTACTAAGCTCGAAATTAAA

[0197] CAR (SEQ ID NO: 5) CD2-TS2-18.1.1-L2H-3033 CAR (SEQ ID NO: 6) CD2-MEDI507 L2H-3043

[0198] ggatccGATATAGTTATGACGCAAGCAGCACCCTCTGTACCTGTGACACCGGGTGAATCCGTTAGTATCTCATGCCGCTCTTCTAAAACCCTCTTGCATTCTAACGGCAATACATATTTGTATTGGTTCCTTCAACGACCAGGACAATCACCGCAAGTGCTTATTTATAGGATGTCTAACTTGGCTAGTGGGGTGCCAAATAGGTTCAGTGGGTCTGGATCTGAGACAACTTTCACGTTGAGAATAAGTAGGGTGGAAGCTGAAGACGTCGGTATATACTACTGTATGCAGCATTTGGAGTACCCTTACACTTTCGGGGGAGGTACTAAGCTCGAAATTAAA ggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAAGTTCAGCTTCAGCAACCAGGTGCTGAATTGGTCCGCCCTGGAACTAGCGTTAAACTGTCTTGTAAGGCATCCGGTTATACGTTTACAAGTTATTGGATGCACTGGATTAAGCAAAGGCCCGAACAAGGCCTTGAATGGATTGGGAGAATTGATCCCTACGATAGCGAGACACACTACAATGAAAAATTTAAAGATAAGGCCATCCTCAGCGTAGATAAGAGCAGTTCTACCGCATACATACAGCTCTCAAGCCTGACGTCAGATGACTCAGCCGTTTATTATTGCTCAAGGCGGGACGCTAAATACGACGGCTATGCGCTTGACTACTGGGGACAAGGCACCACTTTGACAGTCTCCAGTtccgga

[0199] CAR (SEQ ID NO: 7)

[0200] ggatccCAAGTTCAATTGCAGCAACCGGGTGCCGAGTTGGTAAGGCCCGGTGCGTCAGTCAAACTTAGTTGTAAAGCTAGTGGGTACACTTTTACTACGTTCTGGATGAATTGGGTGAAGCAACGACCAGGCCAAGGTCTGGAATGGATCGGCATGATTGACCCGTCTGACTCAGAAGCTCATTACAACCAGATGTTCAAGGACAAGGCGACTCTGACTGTTGATAAAAGCTCAAGCACCGCCTACATGCAGCTCAGTAGCCTCACATCCGAGGATTCCGCAGTGTACTATTGCGCGAGGGGACGAGGGTATGATGACGGCGATGCGATGGACTATTGGGGACAGGGGACCAGCGTAACAGTCAGTAGTggtggcggagggagcggcggtggaggaagcggaggcggaggttccGATATAGTTATGACCCAGTCTCCCGCCTCTCTGGCCGTTAGCTTGGGACAACGCGCTACCATCTCTTACCGAGCGTCTAAGTCCGTCAGTACAAGCGGTTATAGTTACATGCACTGGAACCAGCAAAAGCCCGGACAACCTCCGAGACTCCTGATTTATTTGGTCTCTAACCTTGAGTCAGGTGTCCCAGCCAGATTCTCCGGCTCTGGAAGCGGCACTGACTTTACATTGAACATTCACCCCGTGGAGGAGGAAGACGCTGCTACCTACTATTGCATGCAATTCACGCACTATCCCTACACATTCGGGGGGGGCACGAAATTGGAAATCAAAtccgga

[0201] CD5-17 L2H-3045 CAR (SEQ ID NO: 8)

[0202] ggatccGAGGTTCAGCTTGAGGAGAGTGGGGGAGGTTTGGTAATGCCAGGTGGGTCTTTGAAACTCAGTTGCGCGGCGTCAGGCTTCGCATTTTCCTCCTACGATATGTCCTGGGTCAGACAGACACCCGAGAAGCGGCTGGAATGGGTCGCTTACATTTCCGGGGGAGGATTCACGTACTACCCGGATACAGTAAAGGGGAGATTTACTCTGAGCCGGGACAACGCTAAGAATACCCTCTATCTCCAGATGTCCTCTTTGAAGAGTGAAGACACAGCGATGTATTACTGTGCGAGACAAGGGGCCAATTGGGAGCTGGTTTACTGGGGCCAGGGGACGACATTGACGGTTTCTAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGACATTGTAATGACACAATCACCTGCTACACTTAGCGTGACTCCAGGTGATCGGGTATTCCTGAGCTGCCGCGCATCACAAAGTATATCCGACTTCCTGCACTGGTATCAGCAGAAATCTCACGAAAGTCCCAGGCTGCTGATTAAATACGCTTCCCAGAGTATTAGTGGTATCCCCTCACGATTTTCTGGCAGCGGGAGCGGTAGTGACTTCACTCTTTCTATAAACTCCGTCGAGCCAGAAGACGTGGGGGTGTATCTTTGCCAAAATGGACACAATTTTCCACCAACCTTTGGTGGGGGCACCAAACTCGAAATAAAGtccgga

[0203] CD5-9 H2L-3048 CAR (SEQ ID NO: 9) CD5-9 L2H-3049 CAR (SEQ ID NO: 10)

[0204] ggatccGACATTGTAATGACACAATCACCTGCTACACTTAGCGTGACTCCAGGTGATCGGGTATTCCTGAGCTGCCGCGCATCACAAAGTATATCCGACTTCCTGCACTGGTATCAGCAGAAATCTCACGAAAGTCCCAGGCTGCTGATTAAATACGCTTCCCAGAGTATTAGTGGTATCCCCTCACGATTTTCTGGCAGCGGGAGCGGTAGTGACTTCACTCTTTCTATAAACTCCGTCGAGCCAGAAGACGTGGGGGTGTATCTTTGCCAAAATGGACACAATTTTCCACCAACCTTTGGTGGGGGCACCAAACTCGAAATAAAGggtggcggagggagcggcggtggaggaagcggaggcggaggttccGAGGTTCAGCTTGAGGAGAGTGGGGGAGGTTTGGTAATGCCAGGTGGGTCTTTGAAACTCAGTTGCGCGGCGTCAGGCTTCGCATTTTCCTCCTACGATATGTCCTGGGTCAGACAGACACCCGAGAAGCGGCTGGAATGGGTCGCTTACATTTCCGGGGGAGGATTCACGTACTACCCGGATACAGTAAAGGGGAGATTTACTCTGAGCCGGGACAACGCTAAGAATACCCTCTATCTCCAGATGTCCTCTTTGAAGAGTGAAGACACAGCGATGTATTACTGTGCGAGACAAGGGGCCAATTGGGAGCTGGTTTACTGGGGCCAGGGGACGACATTGACGGTTTCTAGCtccgga

[0205] CD5-34 H2L-3052 CAR (SEQ ID NO: 11) CD5-34 L2H-3053 CAR (SEQ ID NO: 12) CD5-17 H2L-3054 CAR (SEQ ID NO: 13) CD8 Transmembrane Domain Nucleic Acid Sequence (SEQ ID NO: 14):

[0206] ggatccGACGTTGTGATGACGCAAAGTCCCCCGTCACTCCTTGTTACTCTCGGCCAGCCAGCGTCTATCTCTTGCCGGTCAAGCCAGAGCTTGCTCCACTCTAGTGGTAACACGTATTTGAACTGGTTGCTGCAAAGGCCTGGACAATCTCCTCAGCCCCTGATCTATTTGGTTAGCAAACTGGAAAGTGGTGTTCCAGACAGATTTTCAGGGTCTGGATCAGGCACTGATTTCACTCTGAAGATCTCCGGGGTAGAGGCCGAGGACGTGGGAGTCTATTACTGCATGCAGTTTACTCACTATCCTTATACCTTTGGTCAAGGGACGAAACTGGAGATCAAAggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAAGTCCAACTGGTGCAATCAGGCGCAGAAGTCCAACGACCGGGGGCCAGTGTTAAAGTGTCTTGTAAAGCCTCCGGGTACATTTTTACTGAGTACTATATGTACTGGGTCAGACAGGCCCCAGGGCAAGGTTTGGAACTTGTCGGACGCATAGATCCCGAAGACGGTTCTATAGATTACGTTGAGAAGTTCAAAAAGAAAGTCACACTTACTGCGGACACATCTAGTAGCACCGCATATATGGAACTGAGCAGTCTCACCTCAGACGACACCGCAGTGTACTATTGCGCTCGCGGAAAGTTTAACTATAGGTTCGCGTACTGGGGACAGGGGACACTGGTGACTGTTAGCAGCtccgga

[0207] CD8 Transmembrane Domain Amino Acid Sequence (SEQ ID NO: 15): CD8 Hinge Domain Nucleic Acid Sequence (SEQ ID NO: 16): CD8 Hinge Domain Amino Acid Sequence (SEQ ID NO: 17): 4-1BB Nucleic Acid Sequence (SEQ ID NO: 18)

[0208] ggatccAACATTGTACTGACGCAAAGCCCCTCATCTTTGTCTGAGTCACTCGGCGGCAAAGTAACCATCACATGCAAGGCCAGTCAAGACATCAATAAATATATTGCTTGGTATCAGTATAAACCCGGCAAGGGGCCGCGACTGCTGATTCACTACACGAGTACCTTGCAACCGGGCATTCCGAGCCGATTTAGTGGCAGTGGCTCAGGTCGCGATTACTCATTCTCAATAAGTAATCTCGAACCGGAAGACATAGCTACTTATTATTGCTTGCAGTACGATAATTTGTGGACCTTCGGGGGTGGTACAAAGTTGGAAATAAAGggtggcggagggagcggcggtggaggaagcggaggcggaggttccGAGGTCCAACTCGTAGAATCAGGTCCCGGATTGGTGCAACCATCCCAGAGCCTCTCTATTACATGCACGGTCTCTGGATTTAGTCTGACCAATTACGATGTGCATTGGGTGCGCCAGTCTCCCGGCAAGGGGTTGGAATGGCTTGGCGTTATATGGAACTACGGAAATACAGACTATAACGCCGCGTTTATCTCTCGGCTGAGTATACGGAAAGACAGTAGTAAATCCCAGGTCTTTTTTACGATGTCATCCCTGCAAACGCCAGATACCGCAATATATTACTGCGCCAGGAACCACGGTGATGGTTATTATAATTGGTACTTCGATGTGTGGGGTACTGGCACTACAGTCACAGTATCTTCAtctaga

[0209] 4-1BB Amino Acid Sequence (SEQ ID NO: 20) CD3- Amino Acid Sequence (SEQ ID NO: 21): CD2-MEDI507 H2L-3028 CAR Amino Acid Sequence (SEQ ID NO: 25)

[0210] ggatccCAG GTC CAG CTG AAA GAA AGC GGT CCA GAG CTG GAA AAA CCC GGT GCG AGC GTC AAA ATA TCA TGT AAA GCA AGC GGG TAT TCA TTC ACC GCG TAC TCT ATG AAC TGG GTT AAG CAA AAC AAC GGT ATG TCC TTG GAG TGG ATA GGG TCT ATC GAC CCG TAT TAT GGG GAC ACA AAA TAC GCG CAG AAA TTC AAG GGG AAG GCC ACC CTG ACC GTA GAT AAA GCT AGT TCT ACT GCG TAC TTG CAA CTG AAA AGC CTC ACT TCT GAG GAC TCT GCC GTC TAC TAC TGT GCT CGG CGA ATG ATA ACG ACG GGG GAC TGG TAT TTC GAT GTT TGG GGT ACA GGG ACT ACG GTG ACT GTC AGT AGC ggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAT ATC GTC TTG ACT CAA TCA CCT AGT TCT TTG TCT GCG TCC CTT GGC GAC CGA GTC ACC ATA TCT TGC AGA GCG TCA CAG GAC ATT TCAACG TAC CTC AAC TGG TAT CAG CAA AAA CCG GAC GGG ACT GTC AAG CTC TTG ATC TTC TAC ACT TCC AGA CTC CAC GCC GGG GTG CCA AGC AGA TTT AGT GGC TCT GGC AGC GGG ACA CAC CAT AGT CTT ACAATC AGC AAT CTT GAG CAA GAA GAC ATA GCC ACG TAT TTC TGC CAG CAA GGT AAC TCACTT CCG TTC ACG TTT GGT AGT GGC ACC AAA CTG GAG ATA AAA tccgga

[0211] CD2-MEDI507 L2H-3043CAR Amino Acid Sequence (SEQ ID NO: 26) CD2-MEDI507 H2L-3028 scFv Amino Acid Sequence (SEQ ID NO: 27)

[0212] GgatccCAT ATC GTC TTG ACT CAA TCA CCT AGT TCT TTG TCT GCG TCC CTT GGC GAC CGA GTC ACC ATA TCT TGC AGA GCG TCA CAG GAC ATT TCA ACG TAC CTC AAC TGG TAT CAG CAA AAA CCG GAC GGG ACT GTC AAG CTC TTG ATC TTC TAC ACT TCC AGA CTCCAC GCC GGG GTG CCA AGC AGA TTT AGT GGC TCT GGC AGC GGG ACA CAC CAT AGT CTT ACA ATC AGC AAT CTT GAG CAA GAA GAC ATA GCC ACG TAT TTC TGC CAG CAA GGT AAC TCA CTT CCG TTC ACG TTT GGT AGT GGC ACC AAA CTG GAG ATA AAA GGT GGC GGG AGGAGCGGCGGTGGAGGAAGCGGAGGC GGA GGTTC CAG GTC CAG CTG AAA GAA AGC GGT CCA GAG CTG GAAAAAACCC GGT GCG AGC GTC AAA ATA TCA TGT AAA GCA AGC GGG TAT TCA TTC ACC GCG TAC TCT ATG AAC TGG GTT AAG CAA ÷ AAC AAC GGT ATG TCC TTG GAG TGG ATA GGG TCT ATC GAC CCG TAT TAT GGG GAC ACA AAA TAC GCG CAG AAA TTC AAG GGG AAG GCC ACC CTG ACC GTA GAT AAA GCT AGT TCT ACT GCG TAC TTG CAA CTG AAA AGC CTC ACT TCT GAG GAC TCT GCC GTC TAC TAC TGT GCT CGG CGA ATG ATA ACG ACG GGG GAC TGG TAT TTC GAT GTT TGG GGT ACA GGG ACT ACG GTG ACT GTC AGT AGC TCC GGA

[0213] CD2-MEDI507 L2H-3043 scFv Amino Acid Sequence (SEQ ID NO: 28)CD2-MEDI507 VH Amino Acid Sequence (SEQ ID NO: 29)

[0214] ggatccGAGGTTAAACTCGTGGAGAGCGGTGCCGAACTCGTCCGAAGTGGTGCTTCCGTTAAACTCAGTTGTGCCGCGTCAGGATTTAACATAAAAGATTACTACATTCACTGGGTCAAACAGCGCCCGGAGCAGGGGCTTGAATGGATCGGGTGGATTGATCCTGAAAACGGGCGCACCGAATATGCTCCCAAGTTCCAGGGCAAAGCTACTATGACCGCTGACACCTCTAGTAACACTGCCTACCTGCAGTTGAGCTCTCTTACGTCTGAGGATACCGCTGTGTACTACTGTAATAACGGAAATTATGTACGACACTATTACTTCGACTACTGGGGGCAGGGCACTACTGTGACTGTATCTAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGATTGGCTCACACAATCCCCTGCAATCCTGAGTGCATCTCCAGGCGAGAAAGTAACTATGACTTGCAGAGCTATAAGCTCTGTGTCCTACATGCACTGGTATCAGCAGAAGCCAGGTTCTTCCCCGAAGCCGTGGATATATGCTACAAGCAATTTGGCATCCGGTGTTCCCGCCCGGTTTAGTGGCTCCGGTTCTGGGACAAGTTACTCCCTCACGATCAGCAGGGTTGAAGCCGAGGACGCTGCCACTTACTATTGCCAACAGTGGTCAAGTAACCCCAGGACTTTCGGGGGAGGAACTAAACTTGAAATCAAAtctaga

[0215] CD2-MEDI507 VL Amino Acid Sequence (SEQ ID NO: 30)

[0216] GgatccGAT TGG CTC ACA CAA TCC CCT GCA ATC CTG AGT GCA TCT CCA GGC GAG AAA GTA ACT ATG ACT TGC AGA GCT ATA AGC TCT GTG TCC TAC ATG CAC TGG TAT CAG CAG AAG CCA GGT TCT TCC CCG AAG CCG TGG ATA TAT GCT ACA AGC AAT TTG GCA TCC GGT GTT CCC GCC CGG TTT AGT GGC TCC GGT TCT GGG ACA AGT TAC TCC CTC ACG ATC AGC AGG GTT GAA GCC GAG GAC GCT GCC ACT TAC TAT TGC CAA CAG TGG TCA AGT AAC CCC AGG ACT TTC GGG GGA GGA ACT AAA CTT GAA ATC AAA

[0217] GgtggcggagggagcggcggtggaggaagcggaggcggaggttccGAG GTT AAA CTC GTG GAG AGC GGT GCC GAA CTC GTC CGA AGT GGT GCT TCC GTT AAA CTC AGT TGT GCC GCG TCA GGA TTT AAC ATA AAA GAT TAC TAC ATT CAC TGG GTC AAA CAG CGC CCG GAG CAG GGG CTT GAA TGG ATC GGG TGG ATT GAT CCT GAA AAC GGG CGC ACC GAA TAT GCT CCC AAG TTC CAG GGC AAA GCT ACT ATG ACC GCT GAC ACC TCT AGT AAC ACT GCC TAC CTG CAG TTG AGC TCT CTT ACG TCT GAG GAT ACC GCT GTG TAC TAC TGT AAT AAC GGA AAT TAT GTA CGA CAC TAT TAC TTC GAC TAC TGG GGG CAG GGC ACT ACT GTG ACT GTA TCT AGC TCT AGA

[0218] CD2-MEDI507

[0219] ggatccGAGGTCCAACTCGTAGAATCAGGTCCCGGATTGGTGCAACCATCCCAGAGCCTCTCTATTACATGCACGGTCTCTGGATTTAGTCTGACCAATTACGATGTGCATTGGGTGCGCCAGTCTCCCGGCAAGGGGTTGGAATGGCTTGGCGTTATATGGAACTACGGAAATACAGACTATAACGCCGCGTTTATCTCTCGGCTGAGTATACGGAAAGACAGTAGTAAATCCCAGGTCTTTTTTACGATGTCATCCCTGCAAACGCCAGATACCGCAATATATTACTGCGCCAGGAACCACGGTGATGGTTATTATAATTGGTACTTCGATGTGTGGGGTACTGGCACTACAGTCACAGTATCTTCAggtggcggagggagcggcggtggaggaagcggaggcggaggttccAACATTGTACTGACGCAAAGCCCCTCATCTTTGTCTGAGTCACTCGGCGGCAAAGTAACCATCACATGCAAGGCCAGTCAAGACATCAATAAATATATTGCTTGGTATCAGTATAAACCCGGCAAGGGGCCGCGACTGCTGATTCACTACACGAGTACCTTGCAACCGGGCATTCCGAGCCGATTTAGTGGCAGTGGCTCAGGTCGCGATTACTCATTCTCAATAAGTAATCTCGAACCGGAAGACATAGCTACTTATTATTGCTTGCAGTACGATAATTTGTGGACCTTCGGGGGTGGTACAAAGTTGGAAATAAAGtctaga

[0220] HCDR1 (SEQ ID NO: 31) CD2-MEDI507 ​

[0221] atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc

[0222] ​

[0223] IYIWAPLAGTCGVLLLSLVITLYC

[0224] ​ ​ ​

[0225] accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat

[0226] ​ ​ ​

[0227] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0228] ​ ​ ​

[0229] aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg

[0230] CD3-zeta nucleic acid sequence (SEQ ID NO: 19)

[0231] AGAGTGAAGTT CAGCAGGAGC GCAGACGCC CCCGCATACA AGCAGGGCCA GAACCAGCTC TATAACGAGC TCAATCTAGG AC GAAGAGAGG AGTACGATGT TTTGGACAAG AGACGTGGCC GGGACCCTGA GATGGGGGGA AAGCCGAGAA GGAAGAACCC TCAGGAAGGC CTGTACAATG AACTGCAGAA AGATAAGATG GCAGAGGCCT ACAGTGAGAT TGGGATGAA AGGCAGCGCC GGAGGGGCAG GGGGCACGAT GGCCTTTACC AGGTC TCAGTACAG CCACCAAGGA CCCTACGACG CCCTTCACAT GCAGGCCCTG CCCCTCGGC

[0232] ​

[0233] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0234] ​ ​ ​

[0235] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0236] ​

[0237] ​​MALPVTALLLPLALLLHAARPGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0238] ​ ​ ​

[0239] MALPVTALLLPLALLLHAARPGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0240] ​

[0241] GSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKSG

[0242] ​

[0243] GSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSSG

[0244] ​ ​

[0245] QVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSS

[0246] ​

[0247] DVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIK

[0248] ​ ​ ​ ​

[0249] EYYMY

[0250] ​ HCDR2 (SEQ ID NO: 32)

[0251] RIDPEDGSIDYVEKFKK

[0252] CD2-MEDI507 HCDR3 (SEQ ID NO: 33)

[0253] GKFNYRFAY

[0254] CD2-MEDI507 LCDR1 (SEQ ID NO: 34)

[0255] RSSQSLLHSSGNTYLN

[0256] CD2-MEDI507 LCDR2 (SEQ ID NO: 35)

[0257] LVSKLES

[0258] CD2-MEDI507 LCDR3 (SEQ ID NO: 36)

[0259] MQFTHYPYT

[0260] CD2-OKT11 H2L-3029 CAR amino acid sequence (SEQ ID NO: 37)

[0261] MALPVTALLLPLALLLHAARPGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0262] CD2-OKT11 L2H-3030 CAR amino acid sequence (SEQ ID NO: 38)

[0263] MALPVTALLLPLALLLHAARPGSDIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0264] CD2-OKT11 H2L-3029 scFv amino acid sequence (SEQ ID NO: 39)

[0265] GSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIKSG

[0266] CD2-OKT11 L2H-3030 scFv amino acid sequence (SEQ ID NO: 40)

[0267] GSDIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSSSG

[0268] CD2-OKT11 VH amino acid sequence (SEQ ID NO: 41)

[0269] QVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSS

[0270] CD2-OKT11 VL amino acid sequence (SEQ ID NO: 42)

[0271] DIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIK

[0272] CD2-OKT11 HCDR1 (SEQ ID NO: 43)

[0273] SYWMH

[0274] CD2-OKT11 HCDR2 (SEQ ID NO: 44)

[0275] RIDPYDSETHYNEKFKD

[0276] CD2-OKT11 HCDR3 (SEQ ID NO: 45)

[0277] RDAKYDGYALDY

[0278] CD2-OKT11 LCDR1 (SEQ ID NO: 46)

[0279] RSSKTLLHSNGNTYLY

[0280] CD2-OKT11 LCDR2 (SEQ ID NO: 47)

[0281] RMSNLAS

[0282] CD2-OKT11 LCDR3 (SEQ ID NO: 48)

[0283] MQHLEYPYT

[0284] CD2-T11-2-H2L-3031 CAR amino acid sequence (SEQ ID NO: 49)

[0285] MALPVTALLLPLALLLHAARPGSQVQLQQPGAELVRPGASVKLSCKASGYTFTTFWMNWVKQRPGQGLEWIGMIDPSDSEAHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGRGYDDGDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMQFTHYPYTFGGGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0286] CD2-T11-2-H2L-3031 scFv amino acid sequence (SEQ ID NO: 50)

[0287] GSQVQLQQPGAELVRPGASVKLSCKASGYTFTTFWMNWVKQRPGQGLEWIGMIDPSDSEAHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGRGYDDGDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMQFTHYPYTFGGGTKLEIKSG

[0288] CD2-T11-2-H2L-3031 VH amino acid sequence (SEQ ID NO: 51)

[0289] QVQLQQPGAELVRPGASVKLSCKASGYTFTTFWMNWVKQRPGQGLEWIGMIDPSDSEAHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGRGYDDGDAMDYWGQGTSVTVSS

[0290] CD2-T11-2-H2L-3031 VL amino acid sequence (SEQ ID NO: 52)

[0291] DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMQFTHYPYTFGGGTKLEIK

[0292] CD2-T11-2 HCDR1 (SEQ ID NO: 53)

[0293] TFWMN

[0294] CD2-T11-2 HCDR2 (SEQ ID NO: 54)

[0295] MIDPSDSEAHYNQMFKD

[0296] CD2-T11-2 HCDR3 (SEQ ID NO: 55)

[0297] GRGYDDGDAMDY

[0298] CD2-T11-2 LCDR1 (SEQ ID NO: 56)

[0299] RASKSVSTSGYSYMH

[0300] CD2-T11-2 LCDR2 (SEQ ID NO: 57)

[0301] LVSNLES

[0302] CD2-T11-2 LCDR3 (SEQ ID NO: 58)

[0303] MQFTHYPYT

[0304] CD2-TS2-18.1.1-H2L-3032 CAR amino acid sequence (SEQ ID NO: 59)

[0305] MALPVTALLLPLALLLHAARPGSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSGGGGSGGGGSGGGGSGD IVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0306] CD2-TS2-18.1.1-L2H-3033 CAR amino acid sequence (SEQ ID NO: 60)

[0307] MALPVTALLLPLALLLHAARPGSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0308] CD2-TS2-18.1.1-H2L-3032 scFv amino acid sequence (SEQ ID NO: 61) CD2-TS2-18.1.1-L2H-3033 scFv amino acid sequence (SEQ ID NO: 62)

[0309] GSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKSG

[0310] CD2-TS2-18.1.1-L2H-3033 scFv amino acid sequence (SEQ ID NO: 62)

[0311] GSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSSG

[0312] CD2-TS2-18.1.1 VH amino acid sequence (SEQ ID NO: 63)

[0313] EVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSS

[0314] CD2-TS2-18.1.1 VL amino acid sequence (SEQ ID NO: 64)

[0315] DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIK

[0316] CD2-TS2-18.1.1 HCDR1 (SEQ ID NO: 65)

[0317] SYDMS

[0318] CD2-TS2-18.1.1 HCDR2 (SEQ ID NO: 66) CD2-TS2-18.1.1 HCDR3 (SEQ ID NO: 67) CD2-TS2-18.1.1 LCDR1 (SEQ ID NO: 68)

[0319] YISGGGFTYYPDTVKG

[0320] CD2-TS2-18.1.1 LCDR2 (SEQ ID NO: 69) CD2-TS2-18.1.1 LCDR3 (SEQ ID NO: 70)

[0321] QGANWELVY

[0322] CD5-17L2H-3045 CAR amino acid sequence (SEQ ID NO: 71) CD5-17H2L-3054 CAR amino acid sequence (SEQ ID NO: 72) CD5-17L2H-3045 scFv amino acid sequence (SEQ ID NO: 73) CD5-17H2L-3054 scFv amino acid sequence (SEQ ID NO: 74)

[0323] RASQSISDFLH

[0324] CD5-17 VH amino acid sequence (SEQ ID NO: 75) CD5-17 VL amino acid sequence (SEQ ID NO: 76) CD5-9H2L-3048 CAR amino acid sequence (SEQ ID NO: 77) CD5-9L2H-3049 CAR amino acid sequence (SEQ ID NO: 78)

[0325] YASQSIS

[0326] CD5-9H2L-3048 scFv amino acid sequence (SEQ ID NO: 79) CD5-9L2H-3049 scFv amino acid sequence (SEQ ID NO: 80) CD5-9 VH amino acid sequence (SEQ ID NO: 81) CD5-9 VL amino acid sequence (SEQ ID NO: 82)

[0327] QNGHNFPPT

[0328] CD5-9 HCDR1 (SEQ ID NO: 83) CD5-9 HCDR2 (SEQ ID NO: 84)

[0329] MALPVTALLLPLALLLHAARPGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSSRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0330] CD5-9 HCDR3 (SEQ ID NO: 85)

[0331] MALPVTALLLPLALLLHAARPGSEVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIKSRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0332] CD5-9 LCDR1 (SEQ ID NO: 86)

[0333] NIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSS

[0334] CD5-9 LCDR2 (SEQ ID NO: 87)

[0335] EVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIK

[0336] CD5-9 LCDR3 (SEQ ID NO: 88)

[0337] EVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSS

[0338] CD5-34H2L-3052 CAR amino acid sequence (SEQ ID NO: 89) CD5-34L2H-3053 CAR amino acid sequence (SEQ ID NO: 90) CD5-34H2L-3052 scFv amino acid sequence (SEQ ID NO: 91)

[0339] NIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIK

[0340] CD5-34L2H-3053 scFv amino acid sequence (SEQ ID NO: 92) ​

[0341] MALPVTALLLPLALLLHAARPGSQVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSGGGGSHIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0342] ​

[0343] MALPVTALLLPLALLLHAARPGSHIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0344] ​ ​

[0345] GSQVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSHIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKSG

[0346] ​ ​ ​

[0347] HIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSS

[0348] ​ ​

[0349] QVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSS

[0350] ​ ​ ​

[0351] HIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIK

[0352] ​ ​ ​

[0353] AYSMN

[0354] ​

[0355] SIDPYYGDTKYAQKFKG

[0356] ​ ​

[0357] RMITTGDWYFDV

[0358] ​ ​ ​ ​

[0359] RASQDISTYLN

[0360] ​ ​

[0361] YTSRLHA

[0362] ​

[0363] QQGNSLPFT

[0364] ​ ​ ​

[0365] MALPVTALLLPLALLLHAARPGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKSRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0366] ​ ​ ​

[0367] MALPVTALLLPLALLLHAARPGSDWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSSSRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0368] ​ ​ ​ ​

[0369] EVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIK

[0370] ​ ​ ​

[0371] DWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSS

[0372] CD5-34 VH amino acid sequence (SEQ ID NO: 93) CD5-34 VL amino acid sequence (SEQ ID NO: 94)

[0373] EVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSS

[0374] CD5-34 HCDR1 (SEQ ID NO: 95) CD5-34 HCDR2 (SEQ ID NO: 96) CD5-34 HCDR3 (SEQ ID NO: 97) CD5-34 LCDR1 (SEQ ID NO: 98)

[0375] DWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKSR

[0376] CD5-34 LCDR2 (SEQ ID NO: 99) CD5-34 LCDR3 (SEQ ID NO: 100) CRISPR / Cas Introduction of nucleic acids

[0377] DYYIH

[0378] RNA Source of T cells Expansion of T cells

[0379] WIDPENGRTEYAPKFQG

[0380] Pharmaceutical compositions

[0381] GNYVRHYYFDY

[0382] Figure 22 Example 1: New approach to target T cell lymphomas and leukemias without causing T cell toxicity

[0383] RAISSVSYMH

[0384] Figure 1 Figure 2

[0385] ATSNLAS

[0386] Figure 2 Example 2: Anti-CD5 CAR T cells (CART5) and CD5 knockout (KO) normal T cells

[0387] QQWSSNPRT

[0388] Figure 3

[0389] Certain embodiments of the present application include cells that have been modified by a CRISPR / Cas system. CRISPR / Cas systems include, but are not limited to, CRISPR / Cas9 systems and CRISPR / Cpfl systems. In certain embodiments, the present application includes cells that have been modified using a CRISPR / Cas9 system. In certain embodiments, the modification includes knocking out or mutating an endogenous gene, e.g., CD2, CD5, or CD7.

[0390] The CRISPR / Cas9 system is a simple and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a "seed" sequence within the guide RNA (gRNA) and a protospacer adjacent motif (PAM) sequence containing a conserved trinucleotide upstream of the gRNA binding region. By redesigning the gRNAs used in cell lines (such as 293T cells), primary cells, and CAR T cells, the CRISPR / Cas9 system can be engineered to cleave almost any DNA sequence. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suitable for performing multi-gene editing or synergistic activation of target genes.

[0391] One example of a CRISPR / Cas system for inhibiting gene expression, CRISPRi, is described in U.S. Pub. No. US 2014 / 0068797, which is incorporated by reference herein in its entirety. CRISPRi induces permanent gene disruption, which utilizes an RNA-guided Cas9 endonuclease to introduce a DNA double-strand break that triggers error-prone repair pathways, resulting in frameshift mutations. A catalytically dead Cas9 lacks endonuclease activity. When co-expressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcription elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system effectively inhibits expression of a target gene.

[0392] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease is introduced into a cell and forms a complex that is capable of causing a Cas endonuclease to introduce a double-stranded break at the target gene. In certain embodiments, the CRISPR system comprises an expression vector, such as, but not limited to, a pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases can also be used, including, but not limited to, Cpfl, T7, Cas3, Cas8a, Cas8b, CaslOd, Cse1, Csy1, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combination thereof.

[0393] In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector comprises an inducible promoter, such as a promoter that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, e.g., doxycycline). However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition that causes induction of the inducible promoter (e.g., exposure to an agent, such as an antibiotic). This results in expression of the Cas expression vector.

[0394] The guide nucleic acid sequence is specific for a gene and targets the gene for Cas endonuclease-induced double-stranded breaks. The sequence of the guide nucleic acid sequence can be within the locus of the gene. In one embodiment, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more nucleotides in length.

[0395] The guide nucleic acid sequence can be specific for any gene (e.g., CD2, CD5, CD7). The guide nucleic acid sequence comprises an RNA sequence, a DNA sequence, a combination thereof (RNA-DNA combination sequence), or a sequence with synthetic nucleotides. The guide nucleic acid sequence can be a single molecule or a double molecule. In one embodiment, the guide nucleic acid sequence comprises a single guide RNA.

[0396] In the context of forming a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between the target sequence and the guide sequence promotes formation of a CRISPR complex. Perfect complementarity is not necessarily required, so long as there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In certain embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, a target sequence can be within an organelle (e.g., a mitochondrion or nucleus) of a eukaryotic cell. Generally, in the context of a CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of) the target sequence. As with a target sequence, perfect complementarity is not believed to be required, so long as this is sufficient to work. In certain embodiments, a tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of a tracr mate sequence when optimally aligned.

[0397] In other embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system directs formation of a CRISPR complex at one or more target sites. For example, a Cas nuclease, a guide sequence linked to a tracr mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The elements of the CRISPR system combined in a single vector can be arranged in any suitable orientation, such as one element located 5' with respect to ( "upstream of") a second element or 3' with respect to ( "downstream of") a second element. The coding sequence of one element can be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more guide sequences, a tracr mate sequence (optionally operably linked to a guide sequence), and a tracr sequence embedded in one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).

[0398] In certain embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains other than a CRISPR enzyme). The CRISPR enzyme fusion protein can comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Additional domains that can form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In certain embodiments, a labeled CRISPR enzyme is used to identify the location of a target sequence.

[0399] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of the CRISPR system to cells in culture or to host organisms. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles, such as liposomes. Another mode of delivery for CRISPR / Cas9 includes a combination of RNA and purified Cas9 protein, which exists as a Cas9 guide RNA ribonucleoprotein (RNP) complex. (Lin et al., 2014, eLife 3:e04766). Viral vector delivery systems include DNA and RNA viruses, which have episomal or integrated genomes after delivery to cells (Anderson, 1992, Science 256:808-813; and Yu et al., 1994, Gene Therapy 1:13-26).

[0400] In certain embodiments, the CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species. In certain embodiments, the Cas9 can include: spCas9, Cpfl, CasY, CasX, or saCas9.

[0401] Typically, the CRISPR / Cas protein comprises at least one RNA recognition and / or RNA binding domain. The RNA recognition and / or RNA binding domain interacts with the guide RNA. The CRISPR / Cas protein can also comprise a nuclease domain (i.e., a DNAse or RNAse domain), a DNA binding domain, a helicase domain, a RNAse domain, a protein-protein interaction domain, a dimerization domain, and other domains. The CRISPR / Cas protein can be modified to increase nucleic acid binding affinity and / or specificity, to alter enzyme activity, and / or to alter another property of the protein. In certain embodiments, the CRISPR / Cas-like protein of the fusion protein can be derived from a wild-type Cas9 protein or a fragment thereof. In other embodiments, the CRISPR / Cas can be derived from a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, a domain of the Cas9 protein that is not involved in RNA-guided cleavage can be eliminated from the protein, such that the modified Cas9 protein is smaller than the wild-type Cas9 protein. Typically, the Cas9 protein comprises at least two nuclease (i.e., DNAse) domains. For example, the Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC and HNH domains act in concert to cleave single strands, resulting in a double-stranded break in the DNA (Jinek et al., 2012, Science, 337: 816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain). For example, the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., there is no nuclease activity). In some embodiments in which one of the nuclease domains is inactivated, the Cas9-derived protein is capable of introducing a nick into double-stranded nucleic acid (such a protein is referred to as a "nickase"), but does not cleave double-stranded DNA. In any of the above embodiments, any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and / or substitution mutations using well-known methods such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, among other methods known in the art.

[0402] In one non-limiting embodiment, the expression of the CRISPR system is driven by a vector. The art is replete with suitable vectors that can be used in the present application. The vector used is suitable for replication, and optionally integration into a eukaryotic cell. A typical vector contains transcription and translation terminators, an origin of replication, and a promoter to regulate the expression of the desired nucleic acid sequence. The vectors of the present application can also be used in standard gene delivery protocols for nucleic acids. Methods of gene delivery are known in the art (U.S. Patent Nos. 5,399,346, 5,580,859 & 5,589,466, incorporated herein by reference in their entirety).

[0403] Further, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described in, for example, Sambrook et al. (4 th Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012), and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adenovirus-associated viruses, herpes viruses, Sindbis viruses, gammaretroviruses, and lentiviruses. Typically, a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0404] Figures 4-5

[0405] Methods of introducing nucleic acids into cells include physical, biological, and chemical methods. Physical methods of introducing polynucleotides (e.g., RNA) into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Commercially available methods can be used to introduce RNA into target cells, including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM 830 (BTX) (Harvard Instruments, Boston, MA) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). RNA can also be introduced into cells using cationic liposome-mediated lipofection, using polymeric encapsulation, using peptide-mediated transfection, or using biolistic particle delivery systems such as the "gene gun" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

[0406] Biological methods of introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and in particular, retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0407] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in in vitro and in vivo is a liposome (e.g., a man-made membrane vesicle).

[0408] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidyl choline ("DMPC") is available from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY); cholesterol ("Chol") is available from Calbiochem-Behring; dimyristoyl phosphatidyl glycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposomes" is a generic term that encompasses a variety of single and multilamellar lipid carriers formed by creating a closed lipid bilayer or aggregate. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have a series of lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of an aqueous solution, they form themselves into micelles with the lipid bilayer constituting the micelle membrane. The lipid constituents undergo self- reorganization prior to the formation of a closed structure and trap water and dissolved solutes between the lipid bilayers (Ghosh et al., 191 Glycobiology 5; 505-10). However, compositions with different structures in solution as compared to normal vesicular structures are also included. For example, the lipids can assume micellar structures or exist simply as non-uniform aggregates of lipid molecules. Also contemplated are lipofectin- nucleic acid complexes.

[0409] Regardless of the method used to introduce an exogenous nucleic acid into a host cell, or otherwise expose the cell to an inhibitor of the application, to confirm the presence of the nucleic acid in the host cell, a variety of assays can be performed. Such assays include, for example, "molecular biology" assays such as Southern and Northern blots, RT-PCR and PCR; "biochemical" assays such as detection of the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blots) or by assays described herein that recognize agents falling within the scope of the application, known to those of skill in the art.

[0410] In addition, nucleic acids can be introduced by any means, such as transduction of expanded T cells, transfection of expanded T cells, and electroporation of expanded T cells. One nucleic acid can be introduced by one method, while another nucleic acid can be introduced into the T cells by a different method.

[0411] Figure 8

[0412] In one embodiment, the nucleic acid introduced into the T cell is RNA. In another embodiment, the RNA is mRNA comprising in vitro transcribed or synthetic RNA. RNA is produced by in vitro transcription using a template generated using polymerase chain reaction (PCR). DNA from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase for in vitro mRNA synthesis. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable source of DNA. The desired template for in vitro transcription is a chimeric membrane protein. By way of example, the template encodes an antibody, antibody fragment, or a portion of an antibody. By way of another example, the template comprises an extracellular domain comprising a single chain variable domain of an antibody, such as an anti-CD3, and an intracellular domain of a costimulatory molecule. In one embodiment, the template for the RNA chimeric membrane protein encodes a chimeric membrane protein comprising an extracellular domain comprising an antigen binding domain of an antibody derived from a costimulatory molecule, and an intracellular domain derived from a portion of the intracellular domains of CD28 and 4-1BB.

[0413] PCR can be used to generate a template for in vitro transcription of mRNA, which is then introduced into a cell. Methods for performing PCR are well known in the art. Primers for PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for PCR. As used herein, "substantially complementary" refers to a sequence of nucleotides in which a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched, to the intended DNA target. A substantially complementary sequence is capable of annealing or hybridizing to the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify portions of a gene (open reading frame) that is normally transcribed in a cell, including 5' and 3' UTRs. Primers can also be designed to amplify portions of a gene that encode specific domains of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or portions of 5' and 3' UTRs. Primers that can be used for PCR can be generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that is substantially complementary to nucleotides on a DNA template upstream of the DNA sequence to be amplified. As used herein, "upstream" refers to the position 5' with respect to the DNA sequence to be amplified on the coding strand. A "reverse primer" is a primer that contains a region of nucleotides that is substantially complementary to nucleotides downstream of the DNA sequence to be amplified of a double-stranded DNA template. As used herein, "downstream" refers to the position 3' with respect to the DNA sequence to be amplified on the coding strand.

[0414] Chemical structures having the ability to promote stability and / or translational efficiency of the RNA can also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between 0 and 3000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be altered by different methods including, but not limited to, designing primers for PCR that anneal to different regions of the UTR. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths needed to achieve optimal translational efficiency after transfection of the transcribed RNA.

[0415] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be used to modify the stability and / or translational efficiency of the RNA. For example, AU-rich elements in the 3' UTR sequence are known to decrease the stability of the mRNA. Thus, the 3' UTR can be selected or designed based on the properties of the UTR known in the art to increase the stability of the transcribed RNA.

[0416] In one embodiment, the 5' UTR can contain a Kozak sequence of the endogenous gene. Alternatively, when adding a 5' UTR that is not endogenous to the gene of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the translational efficiency of some RNA transcripts, but do not appear to be required for all RNAs to be translated efficiently. The requirement for Kozak sequences for a variety of mRNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of the mRNA.

[0417] In order to be able to synthesize RNA from a DNA template without the need for gene cloning, a transcriptional promoter should be linked to the DNA template upstream of the sequence to be transcribed. When a sequence that will serve as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences for the T7, T3 and SP6 promoters are known in the art.

[0418] In one embodiment, the mRNA has a cap on both the 5' end and the 3' poly(A) tail, which determines ribosome binding, initiation of translation, and stability in the cell mRNA. On a circular DNA template (e.g., plasmid DNA), the RNA polymerase produces long concatameric products, which are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end produces normal size mRNA, which is not efficient in eukaryotic transfection even after post-transcriptional polyadenylation.

[0419] On a linear DNA template, the bacteriophage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0420] A conventional method to incorporate a polyA / T sequence into a DNA template is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often severely contaminated with deletions and other aberrations. This makes the cloning procedure not only laborious and time-consuming, but often unreliable. This is why a method to construct a DNA template with a polyA / T 3' sequence without the need for cloning is highly desirable.

[0421] The polyA / T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail (such as a 100T tail (size can be 50-5000T), or after PCR by any other method including but not limited to DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA and reduces its degradation. In general, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.

[0422] The poly(A) tail of the RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300 to 400 nucleotides results in about a two-fold increase in the efficiency of translation of the RNA. Additionally, attaching different chemical groups to the 3' end can increase mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using a poly(A) tail polymerase. The ATP analogs can further increase the stability of the RNA.

[0423] 5' caps also provide stability to the RNA molecule. In preferred embodiments, the RNA produced by the methods disclosed herein include a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski et al., RNA, 7: 1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0424] The RNA produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent binding of ribosomes to the mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation can be included, which can contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0425] In some embodiments, the RNA, such as in vitro transcribed RNA, is electroporated into cells.

[0426] The disclosed methods can be applied to the modulation of T cell activity in basic research and therapy in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including assessing the ability of genetically modified T cells to kill target cancer cells.

[0427] The methods also provide the ability to control expression levels over a large range by changing, for example, the promoter or the amount of input RNA, so that expression levels can be individually modulated. Furthermore, the PCR-based mRNA production technology greatly facilitates the design of mRNAs with different structures and combinations of their domains.

[0428] One advantage of the RNA transfection method of the present application is that RNA transfection is essentially transient and vector-free. RNA transgenes can be delivered as minimal expressing cassettes without any additional viral sequences to lymphocytes after brief in vitro cell activation and expressed therein. Under these conditions, the probability of transgene integration into the host cell genome is low. Due to the transfection efficiency of RNA and its ability to uniformly modify the entire lymphocyte population, cloning of cells is not necessary.

[0429] Gene modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, which have been successively tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. Various modifications are expected to be used to stabilize IVT-RNA in order to achieve prolonged expression of transferred IVT-RNA.

[0430] Some IVT vectors are known in the literature, which are used as templates for in vitro transcription in a standardized way and have been genetically modified in a way that stable RNA transcripts are generated. The current protocol used in the art is based on plasmid vectors with the following structure: a 5' RNA polymerase promoter, which enables RNA transcription; followed by the gene of interest, which is flanked 3' and / or 5' by untranslated regions (UTRs); and a 3' polyadenylation cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized by a type II restriction enzyme downstream of the polyadenylation cassette (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylation cassette corresponds to the last Poly(A) sequence in the transcript. Due to this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and extend or mask the Poly(A) sequence at the 3' end. It is not clear whether this non-physiological overhang influences the amount of protein produced intracellularly from such constructs.

[0431] RNA has several advantages compared to more traditional plasmid or viral approaches. Gene expression from RNA sources does not require transcription and protein products are rapidly produced after transfection. Further, since RNA can only enter the cytoplasm, but not the nucleus, typical transfection methods result in very high transfection rates. In addition, plasmid-based approaches require that the promoter driving expression of the gene of interest is active in the cells under investigation.

[0432] In another aspect, the RNA construct is delivered into the cell by electroporation. See, for example, formulations and methods for electroporating nucleic acid constructs into mammalian cells as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1. Various parameters are generally known in the relevant research literature as well as in numerous patents and applications in the field, including the electric field strength required for electroporation for any known cell type. See, for example, U.S. Patent No. 6,678,556, U.S. Patent No. 7,171,264, and U.S. Patent No. 7,173,116. Devices for therapeutic applications of electroporation are commercially available, for example, the MedPulser TM DNA electroporation therapy system (Inovio / Genetronics, San Diego, Calif.), and described in patents such as U.S. Patent No. 6,567,694; U.S. Patent No. 6,516,223, U.S. Patent No. 5,993,434, U.S. Patent No. 6,181,964, U.S. Patent No. 6,241,701, and U.S. Patent No. 6,233,482; electroporation can also be used to transfect cells in vitro, as described, for example, in US20070128708A1. Electroporation can also be used to deliver nucleic acids into cells in vitro. Thus, the electroporation-mediated administration of nucleic acids including expression constructs into cells provides an exciting new means for delivering RNA of interest to target cells, utilizing any of a variety of available devices and electroporation systems known to those skilled in the art.

[0433] Figure 8

[0434] In certain embodiments, a source of T cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain embodiments, any number of T cell lines available in the art can be used. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis or leukapheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium, such as phosphate buffered saline (PBS), or a wash lacking calcium and possibly lacking magnesium or possibly lacking many, if not all, divalent cations, for subsequent processing steps. After washing, the cells can be resuspended in a variety of biocompatible buffers, for example, Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0435] In another embodiment, T cells are isolated from peripheral blood, for example, by PERCOLL TM gradient centrifugation, by lysing red blood cells and depleting mononuclear cells. Alternatively, T cells can be isolated from umbilical cord. In any case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0436] Umbilical cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including but not limited to CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using isolated antibodies, biological samples containing antibodies (such as ascites), antibodies bound to a physical support, and cell-bound antibodies.

[0437] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed against surface markers unique to the cells being negatively selected. The preferred method is by negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the cells being negatively selected for cell sorting and / or selection. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies generally includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.

[0438] To isolate a desired cell population by positive or negative selection, the concentration of cells and surface (e.g., particles, such as beads) can be varied. In certain embodiments, it can be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of the cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In further embodiments, greater than 100 million cells / ml is used. In further embodiments, a cell concentration of 100, 150, 200, 250, 300, 350, 400, 450, or 500 million cells / ml is used. In yet further embodiments, a cell concentration of 750, 800, 850, 900, 950, or 1000 million cells / ml is used. In further embodiments, a concentration of 125 or 150 million cells / ml can be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0439] T cells can also be frozen after the washing step, which does not require the monocyte removal step. While not wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and some degree of monocytes from the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While a variety of freezing solutions and parameters are known in the art and will be useful in context, in a non-limiting example, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen at a rate of 1 °C per minute to -80 °C and stored in the gas phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used, as well as uncontrolled freezing at -20 °C immediately or in liquid nitrogen.

[0440] In one embodiment, the T cell population is contained within a cell, such as a peripheral blood mononuclear cell, umbilical cord blood cell, purified T cell population, and T cell line. In another embodiment, the peripheral blood mononuclear cell comprises the T cell population. In yet another embodiment, the purified T cell comprises the T cell population.

[0441] Figure 13

[0442] In certain embodiments, the T cells disclosed herein can be expanded by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold or more, and any and all whole or partial integers therebetween. In one embodiment, the T cells are expanded in a range of between about 20-fold and about 50-fold.

[0443] After culturing, the T cells can be incubated in the culture medium in the culture device for a period of time, or until the cells reach confluence or high cell density for optimal passaging, before the cells are passed to another culture device. The culture device can be any culture device typically used for culturing cells in vitro. Preferably, the confluence level is 70% or greater before the cells are passed to another culture device. More preferably, the confluence level is 90% or greater. The period of time can be any time suitable for in vitro cell culturing. The T cell culture medium can be replaced at any time during the culturing of the T cells. Preferably, the T cell culture medium is replaced approximately every 2-3 days. The T cells are then harvested from the culture device, which can be used immediately thereafter or cryopreserved for later use. In one embodiment, the present application includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing the nucleic acid into the T cells.

[0444] In another embodiment, the method includes isolating T cells and expanding the T cells. In another embodiment, the present application further includes cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding a chimeric membrane protein.

[0445] Another procedure for expanding cells ex vivo is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). Expansion (as described in U.S. Patent No. 5,199,942) can be in addition to or in place of the other expansion methods described herein. Briefly, ex vivo culturing and expansion of T cells includes the addition of cell growth factors (such as those described in U.S. Patent No. 5,199,942 or other factors such as flt3-L, IL-1, IL-3, and c-kit ligand). In one embodiment, expanding the T cells includes culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0446] The culturing steps described herein (following contact with the reagents described herein or electroporation) can be very short, for example less than 24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing steps as further described herein (contact with the reagents described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0447] Various terms are used to describe cells in culture. Cell culture generally refers to cells taken from a living organism and grown under controlled conditions. Primary cell culture is the culture of cells, tissues, or organs taken directly from an organism and prior to the first subculture. Cells expand in culture when placed in growth medium under conditions that promote cell growth and / or division, resulting in a larger population of cells. When cells expand in culture, the rate of cell proliferation is generally measured by the amount of time it takes for the cell population to double in size, also known as the doubling time.

[0448] Each round of subculturing is referred to as a passage. When cells are subcultured, they are said to have been passaged. A particular population or cell line is sometimes referred to or characterized by the number of times it has been passaged. For example, a population of cells in culture that has been passaged 10 times can be referred to as a P10 culture. A primary culture (i.e., the first culture after cells are isolated from a tissue) is referred to as P0. After the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. Those skilled in the art will appreciate that there can be multiple population doublings during a passage; therefore, the number of population doublings of a culture is greater than the number of passages. During the period between passages, the expansion of cells (i.e., the number of population doublings) depends on multiple factors, including but not limited to seeding density, substrate, medium, and time between passages.

[0449] In one embodiment, cells can be cultured for a number of hours (about 3 hours) to about 14 days or any whole number of hours therebetween. Conditions suitable for T cell culture include appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 15, (Lonza)), which can contain factors required for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-a or any other additives known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either without serum or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures, not in cell cultures to be infused into a subject. Target cells are maintained under conditions required to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0450] Media for culturing T cells can include agents that can co-stimulate T cells. For example, an agent that can stimulate CD3 is an antibody against CD3, and an agent that can stimulate CD28 is an antibody against CD28. This is because, as demonstrated by the data disclosed herein, cells isolated by the methods disclosed herein can expand approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more. In one embodiment, T cells expand in the range of about 20-fold to about 50-fold or more by culturing the electroporated population.

[0451] In one embodiment, the method of expanding T cells can further comprise isolating the expanded T cells for further application. In another embodiment, the method of expansion can further comprise subsequent electroporation of the expanded T cells, followed by culturing. The subsequent electroporation can comprise introducing a nucleic acid encoding an agent into the population of expanded T cells, such as transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with the nucleic acid, wherein the agent further stimulates the T cells. The agent can stimulate the T cells, such as by stimulating further expansion, effector function, or another T cell function.

[0452] Figure 13

[0453] Pharmaceutical compositions of the application can comprise modified cells or cell populations as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers such as neutral buffered saline, sulfate -buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the application are preferably formulated for intravenous administration.

[0454] Pharmaceutical compositions of the application can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease - although appropriate dosages can be determined by clinical trials.

[0455] With respect to a subject receiving therapy, the cells of the application to be administered can be autologous, allogeneic, or xenogeneic.

[0456] Cells of the application can be administered in dosages and by routes and at times to be determined in appropriate preclinical and clinical experiments and trials. Cell compositions can be administered multiple times at dosages within these ranges. Administration of cells of the application can be combined with other methods for treating the desired disease or condition as determined by those skilled in the art.

[0457] It can generally be stated that pharmaceutical compositions comprising the T cells described herein can be administered at a dose of 10 4 to 10 9 cells / kg body weight, in some cases 10 5 to 10 6The T cell compositions can be administered in a dose of 1 x 106cells / kg body weight, including all integer values within that range. The T cell compositions can also be administered multiple times at these doses. The cells can be administered by using infusion techniques well known in the art of immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by monitoring the patient for signs of disease and adjusting the treatment accordingly by those skilled in the medical arts.

[0458] Administration of the modified cells of the present application can be carried out in any convenient manner known to those of skill in the art. The cells of the present application can be administered to a subject by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be injected or administered intraperitoneally to a patient via an artery, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (i.v.), or intraperitoneally. In other cases, the cells of the present application are injected directly into an inflammatory site of a subject, a local disease site of a subject, a lymph node, an organ, a tumor, and the like.

[0459] It is understood that the methods and compositions useful in the present application are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells of the present application, methods of expansion and culture, and methods of treatment, and are not intended to limit the scope of what the inventors regard as their application.

[0460] The practice of the present application employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the application, and thus, can be considered in making and practicing the application. Particularly useful techniques for particular embodiments will be discussed in the following sections.

[0461] Experimental Examples

[0462] The application will now be described with reference to the following examples. These examples are provided for purposes of illustration only and the application is not limited to these examples, but encompasses all variations that are evident as a result of the teaching provided herein.

[0463] The materials and methods used in these experiments are now described.

[0464]

[0465] sgRNAs were designed to target CD2, CD5, or CD7 (e.g., SEQ ID NO: 22 to 24, respectively) and synthesized using the GeneArt Precision sgRNA Synthesis Kit. A Cas9 expression plasmid (pGEM-Cas9) was amplified and linearized. Cas9 RNA was synthesized using the mMessage mMachine T7 Ultra Kit. CRISPR editing was performed in Jurkat cells: CD2 / CD5 / CD7 sgRNAs and Cas9 were transfected into Jurkat cells via electroporation. CD2 / CD5 / CD7 expression on Jurkat cells was detected by flow cytometry, and the most efficient CD2 / CD5 / CD7 sgRNAs were identified. CRISPR editing was then performed in primary human T cells using the most efficient CD2 / CD5 / CD7 sgRNAs: the selected sgRNAs and Cas9 RNA were electroporated into primary human T cells. CD2 / CD5 / CD7 expression on primary human T cells was detected by flow cytometry to validate knockout / editing efficiency.

[0466] Specifically, fresh CD4 / CD8 T cells were obtained on day 0 and incubated with dynabeads. On day 4, the cells were debeaded and then electroporated with Cas9 and sgRNA. Conditioned medium (TCM (X-vivo15, 5% human serum, glutamine), IL-7 10 ng / ml and IL-15 10 ng / ml) was added to the cells. On day 6, the cells were transduced with CAR lentivirus. On day 9, CAR expression was assessed. When the volume was <300 fl, the cells were fed to 0.8 e6 / ml and frozen. Figure 26 ).

[0467] CAR constructs: All constructs were generated using the lentivirus pTRPE 4-1BB CD3ζ backbone. OKT11 CAR and TS2 / 18.1.1 CAR were constructed using scFv derived from hybridomas purchased from ATCC (respectively...). CRL-8027 TM and HB-195 TM The T11-2 CAR was constructed using an scFv derived from an antibody produced from a hybridoma received from Ellis Reinherz. All CD5 CARs were constructed using scFvs of antibody sequences disclosed in WO2010 / 022737A1—the contents of which are incorporated herein by reference in their entirety.

[0468] The results of the experiment will now be described.

[0469] Figure 26

[0470] T-cell lymphomas and leukemias generally have a very poor prognosis, and there are few available treatment options for these patients. Chimeric antigen receptor T-cell (CART) immunotherapy has achieved unprecedented results in CD19+ B-cell non-Hodgkin lymphoma (B-NHL). In this article, another successful “CART19-like” product was designed to target T-NHL. Since CD19 is not expressed in T-NHL, other targets such as CD2, CD5, and CD7 were evaluated for CARART therapy. However, all of these targets are also expressed by normal T cells, leading to unacceptable clinical toxicities (T-cell hypoplasia—immunodeficiency). Example 3: Anti-CD2 CAR T cells (CART2) and CD2 knockout (KO) normal T cells In this paper, a safe and effective CAR-T strategy for treating T-cell lymphoma was developed by editing normal T cells resistant to CAR-T killing. Figure 3 CAR-T therapy is feasible for T-cell lymphomas and leukemia when CAR-T targets (CD2, CD5, CD7) are temporarily removed from normal T cells, thus avoiding CAR-T-mediated killing and immunodeficiency. Figure 4 ).

[0471] Figure 7

[0472] This article discloses a two-pronged immunotherapy approach, comprising anti-CD5 CAR T cells (CART5) and CD5 knockout (KO) normal T cells (CART5). Figure 7 CART5 destroys T-cell lymphoma (e.g., T-NHL) or T-cell leukemia cells, but it also kills normal T cells. Infusion of CD5 KO normal T cells provides CART-resistant T-cell immunity until CART5 cells are depleted, in some cases by using suicide genes (e.g., iCasp9, CD20 / rituximab, or others).

[0473] CD5 was selected as a target for T-NHL due to its high expression on T-NHL cells and its absence in other tissues except for T cells and a small subset of B cells. Six anti-CD5 CAR constructs were generated using single-stranded variable fragments (scFv) with different affinities (high, medium, and low affinity, #17, #34, and #9, respectively, Klitgaard JL, et al. (2013) British Journal of Haematology 163:182-93)) and expressed on T cells. Figure 7). Interestingly, while CD5 is expressed in 100% of CART cells, anti-CD5 CARTs do not require CD5 knock-out to make CART5 cells (Mamonkin M, et al. (2015) Blood 126:983-92), although CD5 expression is lower compared to control T cells Figure 14 ) In the absence of CD5 CRISPR-Cas9 KO, CD5 mean fluorescence intensity (MFI) in CART5 is 10-fold lower compared to control T cells, while another pan T cell marker like CD2 is unchanged Figure 9

[0474] In vitro and in vivo activity of different CART5 constructs was compared. Construct C3054 from high affinity scFv #17 showed the best in vivo killing. Jurkat cells were transduced with different CAR5 constructs Example 4: CART2 and CART5 testing targeting epitopes and affinities shown on the left) and with GFP-NFAT reporter, then co-cultured with CD5+tumor cells (or controls) for 24 hours. Lead CART5 (C3054) showed increased NFAT activation Figure 6

[0475] Lead anti-CD5 CARTs were implemented using a suicide system and their function was tested in vitro and in vivo. Without wishing to be bound by a particular theory, removal of CD5 (CRISPR-Cas9 KO) further increases CART5 anti-tumor effect by eliminating possible cis surface interactions between CD5 and CAR5 on CART5.

[0476] Insertion of suicide system in lead CART5 product: lead candidate CART5 (C3054) product was engineered to express a suicide system Figure 10 ). A P2A bi-cistronic vector encoding CAR5 and inducible-caspase9 suicide system was developed (DiStasi A, et al. (2011) 365: 1673-83) (iCART5). Two orientations, CAR5-P2A-iCasp9 and iCasp9-P2A-CAR5 Figure 11 were cloned to define the most efficient one (higher percentage of bi-expressing cells and lower CAR5+iCasp9-% to ensure safety). The clinical grade compound rimiducid (AP1903, Bellicum Pharmaceuticals) was used to efficiently eliminate iCART5.

[0477] ​​In vitro testing of iCART5: Newly generated iCART5 were compared to WT CART5 to confirm their efficacy (luciferase-based in vitro killing) and phenotype / functionality upon antigen stimulation (CD5+ Jurkat T leukemia cells) (flow cytometry phenotype, 30-plex cytokine analysis by Luminex assay, CFSE proliferation and CD 107a degranulation). Importantly, in vitro depletion was tested by co-culturing iCART5 with different concentrations of rimiducid (0, 0.03, 0.3, 3, 10 nM) and checking killing at 15', 30' and 2, 6, 12 and 24 hours. iCART5 were also tested against primary T-NHL cells by a killing assay established using primary CFSE labeled Sezary cells.

[0478] In vivo testing of iCART5: The xenograft model in vivo (Ruella M, et al. (2016) J Clin Invest) [using click beetle green (CBG) + T-leukemia cell line Jurkat cell line and click beetle red (CBR) + iCART5] was used to test the ability of rimiducid (50 ug / mouse 23) to deplete iCART5 vs. WT CART5 in vivo. NOD SCID gamma-deficient mice (NSG) mice (8 mice per group) were injected with 2x10e6 iCART5 or WT CART5 cells / mouse. Tumor burden was assessed as bioluminescence (CBG) over time and T cell phenotype was investigated by flow cytometry and expansion by bioluminescence (CBR) at multiple time points (hours / days). Mice were kept long term (3-4 months) for survival and monitoring of relapse. The human T-NHL xenograft model was previously established by i.v. injection of primary Sezary cells. This model was used to test the anti-tumor and depletion efficacy of iCART5. 6 CART5 cells / mouse. Tumor burden was assessed as bioluminescence (CBG) over time and T cell phenotype was investigated by flow cytometry and expansion by bioluminescence (CBR) at multiple time points (hours / days). Mice were kept long term (3-4 months) for survival and monitoring of relapse. The human T-NHL xenograft model was previously established by i.v. injection of primary Sezary cells. This model was used to test the anti-tumor and depletion efficacy of iCART5.

[0479] Evaluation of CD5 KO in CART5: Preliminary data suggest that CD5 CART5 is more potent than WT CART5 in vivo. CD5 is knocked out on CART5 cells using CRISPR-Cas9. The CRISPR-Cas9 CART expansion protocol was previously optimized. Wild type CART5 is compared to CD5 KO CART5 in vitro by testing CART5 viability, antigen-driven proliferation (using CFSE labeling), cytokine production (by 30-plex Luminex), degranulation (CD 107a assessment by flow cytometry), cytotoxicity (luciferase-based), and phenotype (memory subsets, Thl / Th2). Cell lines (e.g., Jurkat) and primary samples are both used as targets. In vivo comparison of CD5 KO vs. WT CART5 (1 x 10e6 cells / mouse) is performed in Jurkat-bearing NSG mice and monitored for expansion and phenotype in peripheral blood at day 10 and day 14.

[0480] Mechanism of enhanced CART5 function by CD5 KO: CD5 KO has been shown to improve CART5 activity, additional studies are performed to understand the mechanism: i. confocal imaging to analyze the localization of CAR5 and CD5 on CART5 cells; ii. single molecule imaging (ONI nanoimager) demonstrates that CAR5 binds CD5 in cis; iii. expression of CAR5 in CD5+ Jurkat and shows that CART5 fails to kill CAR5+ Jurkat because the CD5 epitope is masked by CAR5; and iv. since CD5 has an inhibitory effect on T cell activation, we will study the activation of CART5 (phospho-flow cytometry) in the presence or absence of CD5.

[0481] Generation of CART-resistant normal T cells to avoid T cell aplasia: CART5 cannot distinguish between neoplastic T cells and normal T cells because both express similar levels of CD5. CART-resistant normal T cells are developed to be co-infused with anti-T-NHL CART to ensure immunity during CART anti-tumor activity. CRISPR-Cas9 gene editing is used to knock out CD5 in normal T cells, making them invisible to CART5. An optimized CART expansion protocol is used to generate a highly efficient CRISPR-Cas9 gRNA (#4) that can KO ~95% of CD5 in normal T cells. Data show that CD5 KO T cells are resistant to CART5, while WT T cells (CD5+) are potently killed within 24 hours.

[0482] Manufacture of CD5 KO normal T cells: CD5 KO normal T cells were developed using a high efficiency gRNA (#4) used with Cas9 protein (ThermoFisher v2) electroporated with Lonza 4D Nucleofector. On day 1, CRISPR-Cas9 KO was performed and then cells were cultured for 2 days at 30°C to increase gene editing, then activated with anti-CD3 / CD28 Dynabeads (beads: 1 T cell) and expanded until they reached a cell volume < 300 fl.

[0483] In vitro evaluation of resistance of CD5 KO normal T cells to iCART5 killing:

[0484] Resistance of CD5 KO normal T cells to CART5 was tested in vitro by performing a killing assay (CFSE labeling of target T cells). Preliminary results show that CD5 KO confers resistance. Three additional T cell donors will be tested.

[0485] In vivo evaluation of resistance of CD5 KO normal T cells to iCART5 in a xenogeneic implantation model: NSG mice (8 mice / group) were implanted with luciferase+ CD5 KO normal T cells or WT and two days later injected with autologous iCART5. The effect of iCART5 on CD5 ko and WT normal T cells was evaluated by bioluminescence. Once the WT T cells were completely eliminated by iCART5 (luminescence), rimiducid was administered to deplete iCART5. WT T cells were then re-injected to demonstrate that normal T cells can repopulate the host. Mice were bled weekly to evaluate CART expansion.

[0486] Evaluation of the effect of CD5 KO on normal T cell function: The effect of CD5 KO in normal T cells was studied by carefully investigating T cell effector functions. Cytokine production (30-plex Luminex), proliferation (CFSE) and activation of CD5 KO T cells vs. WT were measured after TCR specific stimulation (anti-CD3 / CD28 beads). It was also tested whether CD5 KO T cells proliferate and produce similarly to WT upon exposure to common infections.

[0487] Definition of the optimal CD5 KO normal T cell dose for clinical use: To ensure enough T cell immunity against the most common infections, while using in silico and experimental approaches (TCR sequencing and tetramer staining of TCRs specific for infectious agents) to define the minimum number of cells to be infused in relapsed or refractory (r / r) T-NHL patients.

[0488] In a phase 1 pilot clinical trial in patients with advanced T-cell lymphoma, the simultaneous infusion of iCART5 and CD5 KO normal T cells was tested: an Investigational New Drug (IND) package was developed and submitted to the FDA. A phase 1 clinical trial was initiated to test the anti-T-NHL CART approach in patients. The IND package was based on preliminary results and further data from the experiments described herein.

[0489] Clinical trial protocol design: The phase 1 clinical trial included r / r T-NHL patients treated using a 3+3 protocol design. From one apheresis, two products were generated using enriched T cells: #1. CRISPR-Cas9 CD5 KO normal T cells and #2. iCART5. The first product to be infused was the KO normal T cells, followed by iCART5 the next day. The first patient cohort received lymphodepletion [cyclophosphamide (60 mg / kg x 2 days) and fludarabine (25 mg / m2x 5 days)] and product #1. If no dose limiting toxicity (DLT) was observed, cohort 2 received lymphodepletion, product #1 and 1-5 x 10e 7 Total iCART5 (product #2) (10%, 30%, 60% of total dose); 1-5 x 10e 7 Total CART5 is based on the suboptimal dose of CART19 experiment in B-NHL8. If no DLT was observed in cohort 2, cohort 3 received lymphodepletion, product #1 and 1-5 x 10e 8 Total CART5 (full dose). If no toxicity was observed within the first 4 weeks, patients from cohort 1# were allowed to enter cohort 2#. iCART5 cells were depleted using the dimerizing agent rimiducid (NCT02744287) to prevent possible long-term T cell toxicity, depending on tumor clearance rate (and maximum reached at month 6).

[0490] IND package preparation and FDA submission: Clinical grade manufacturing was optimized in collaboration with the Clinical Vaccine and Cell Production Facility (CVPF). Results from all described preclinical experiments were formatted together with the clinical trial protocol to fit the IND application. Extensive support for the preparation of the IND was provided within the CCI and ACC.

[0491] Patient enrollment and treatment: After successful IND submission and approval from all regulatory agencies, a phase 1 trial within the Lymphoma Program at the University of Pennsylvania began (Principal Investigator: Dr. Stephen Schuster; Scientific Director: Dr. Marco Ruella). The Lymphoma Program has a dedicated clinical research unit (CRU) with extensive experience in managing early-stage research. Dr. Ruella is the principal investigator for the trial, and Dr. Carl June is the scientific protocol advisor. Manufacturing of both products is performed at the CVPF.

[0492] Related studies: Patient samples (peripheral blood) are analyzed at multiple time points (apheresis, days 1, 0, 7, 14, 28, 60, 90) to test CART expansion (qPCR and flow cytometry), CART phenotype (CyTOF), CART gene expression profile (GEP) (NanoString, single-cell RNAseq 10X Genomics), and cytokine levels in serum (Luminex, 30-plex array). Additional studies are performed on tumor biopsies pre- and post-treatment (RNAseq and Hyperion analysis of tumor microenvironment) when available.

[0493] This trial will be an important milestone in the development of novel combination immunotherapy, as it represents an innovative immunotherapy for treating T-cell non-Hodgkin lymphoma that avoids toxicity. Anti-CD5 CAR T cells kill tumor T cells but, due to similar CD5 expression, also inevitably kill normal T cells. However, the strategy described herein includes co-infusion of normal T cells with CD5 knocked out, thus ensuring T cell immune protection during the anti-tumor activity of CAR T5. CAR T5 cells are then depleted using a suicide system to ensure long-term normal immune reconstitution. This is one of the first CAR T trials for T-NHL and is also the only one that includes a two-pronged approach to address toxicity issues. Prognosis for T-NHL is poor, and there are currently no active immunotherapies available. Therefore, the development of this innovative strategy represents a vertical advance in the fields of hematology and immunotherapy. Based on the clinical results of the phase 1 trial and the results of the related studies, the strategy can also be applied simultaneously to target multiple targets to avoid antigen-loss escape (e.g., CAR T5 + CAR T7) or to combine CAR T with small molecules that can enhance CAR T-mediated killing.

[0494] Figure 15

[0495] Disclosed herein is a two-pronged immunotherapy method that includes anti-CD2 CAR T cells (CAR T2) and CD2 knockout (KO) normal T cells (Figure 15 CART2 destroys T-cell lymphoma (e.g., T-NHL) or T-cell leukemia cells, but it also kills normal T cells. Infusion of CD2 KO normal T cells provides CART-resistant T-cell immunity until CART2 cells are depleted, in some cases by using suicide genes (e.g., iCasp9).

[0496] Guide RNAs were engineered to knock out the CD2 gene (and CD5) using the CRISPR / Cas9 system. CD2 was effectively knocked out in 78% of the T cell population. This resulted in the generation of second-generation anti-CD2 and anti-CD5 CARs (CART2 and CART5, respectively). Figure 12 Knockout cells (CD2KO and CD5KO) were incubated with their corresponding CART cells (CART2 and CART5, respectively), stimulated, and population doubling was measured. Figure 16 Electroporated cells without gRNA (Mock) were used as a comparative control. Without CD2 KO, CART2 cells would not expand (…). Figure 17 With KO CART2 and CART5, approximately 5 to 8 population doublings were achieved. Figure 17 ).

[0497] Jurkat cells were transduced with different CAR2 constructs and the GFP-NFAT reporter gene, and then co-cultured with CD2+ tumor cells (or controls) for 24 hours. Lead CAR2 (C3043) showed increased NFAT activation. Figure 17 ).

[0498] The CART amplification protocol was optimized, allowing CART2 and CART5 cells to continue expanding for up to 18 days when incubated with CD2KO or CD5KO cells. Figure 17 ).

[0499] Figure 18

[0500] Figure 18 The manufacturing process of CD5 (or CD2, or CD7) KO and the efficiency of CRISPR-Cas9 KO are illustrated.

[0501] Six different CAR2 and six CAR5 constructs were challenged in vitro by co-culturing them with luciferase-enhanced Jurkat cells (a T-cell leukemia cell line). At 24 hours, total killing was measured as a relative reduction in luminescence. For CAR2, only #3029, #3030, and #3043 showed antitumor activity. Example 5: Anti-CD7 CAR T cells (CART7) and CD7 knockout (KO) normal T cells For CART5, all constructs exhibited antitumor activity. Figure 25Leader CART candidates (CART2 C3043 and CART5 C3054) were selected and tested. The effect of CD2 or CD5 knockout on CART function was tested. CART2- and CART5-resistant T cells were successfully generated (CD5 and CD2 were knocked out in normal T cells).

[0502] The activity of CART2 and CART5 against cutaneous T-cell lymphoma was tested. A 24-hour killing assay was performed. CART2 cells were compared with primary Sezary cells (leukemia-associated cutaneous T-cell lymphoma) and HH Sezary cell line (…). Figure 25 CART5 is active. CART5 is also active against HH cells. Example 6: Bispecific CAR T cells ).

[0503] The in vivo efficacy of CART2 and CART5 was measured. NSG mice were implanted with luciferase + Jurkat cells, and mice were randomly assigned on day 7 to receive either control T cells or CART2 or CART5 (1x10⁻¹²). 6 Mice were imaged weekly using IVIS Xenogen Spectrum and analyzed using LivingImage software. CART2 C3043 and CART5 C3054 were the most effective. Figure 19 ).

[0504] CART2 and CART5 have been shown to recognize and kill normal T cells (both autologous and allogeneic). Figures 20A-20B ).

[0505] It was also demonstrated that the removal of CAR targets protects normal T cells from CAR-T killing. Example 6: CD5 KO enhances CART immunotherapy CD5 KO, but not WT, normal T cells are resistant to CART5 killing. Normal resting T cells are killed by CART2 ( Figure 21 (top) and CART5 ( Figure 21 (Bottom) Recognizes and kills. Effective knockout of CD2 or CD5 from normal T cells using CRISPR-Cas9 resulted in resistance to CART2 or CART5 killing, respectively. Figure 22 ).

[0506] CMV-specific T cells are present in the products of CD2KO and CD5KO normal T cells. Figure 22 CD2 and CD5 KO normal T cells maintain the ability to recognize CMV peptides and produce cytokines. Figures 23A-23B; HLA-A-02:01 - CMV PP65 NLVPMVATV dextran (SEQ ID NO: 101); ICS after 4 hours of exposure to CETF peptide. After secondary culture with CMV-peptide pulsed APCs.

[0507] Figure 24

[0508] Disclosed herein are dual immunotherapy approaches that include anti-CD7 CAR T cells (CART7) and CD7 knockout (KO) normal T cells. CART7 destroy T cell lymphoma (e.g., T-NHL) or T cell leukemia cells, but also kill normal T cells. Infusion of CD7 KO normal T cells provides CART-resistant T cell immunity until the CART7 cells are exhausted, in some cases through the use of a suicide gene (e.g., iCasp9).

[0509] Guide RNAs were designed to knockout the CD7 gene using the CRISPR / Cas9 system. CD7 was effectively knocked out in 79% of the T cell population Other embodiments ). Six anti-CD7 CARs were generated ​ ).

[0510] ​

[0511] Two lentivirus constructs were generated that include CAR5 (C3054) and CAR2 (C3043) linked by a P2A sequence ​ ). Gene expression is driven by an EFla promoter. The CAR5 construct has a 4-1BB costimulatory domain and a CD3 zeta signaling domain. Effective knockout of CD2 and CD5 in normal T cells was demonstrated ​ ).

[0512] ​

[0513] CD5 KO CART5 was shown to be more effective than CD5+ CART5 in vivo. CD5 KO increased the anti-tumor efficacy of CART5 ​ ). CD5 KO CART5 (2 x 10 6 cells / mouse) resulted in complete long-term responses and longer survival compared to WT CART5 in a Jurkat T-ALL xenograft model using NSG mice ​ ).

[0514] CD5 KO CART19 was also more effective than CD5+ CART19 in vivo. CD5 KO increased the anti-tumor efficacy of CART19 ​). CD5 KO CART 19 demonstrated significantly higher tumor control compared to WT CART 19 in the NALM6 B-ALL xenograft model ( ​ ) in vivo.

[0515] CART5 and CART2 were also able to target 20% of AML. CART2 cells were co-cultured with CD2+ AML cells and showed significant killing at 24 hours ( ​ ) in vitro.

[0516] CART5 also targeted 100% of CLL and MCL. Cytotoxicity assays were performed and demonstrated that CART5 cells can recognize and kill CD5+ MCL cell lines (Jeko-1 and Mino) ​ ) in vitro.

[0517] These data suggest that knocking out CD5 enhances CART therapy when treated with anti-CD5 CAR, or surprisingly when treated with a different CAR T cell, such as CD 19 CART cells.

[0518] ​

[0519] The recitation of elements in any definition of a variable in this document includes a definition of the variable as any single element or combination (or sub-combination) of listed elements. The recitation of embodiments of this document includes an embodiment as any single embodiment or combination of any other embodiment or portions thereof.

[0520] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. Although specific embodiments of the present application have been disclosed, it is to be understood that other embodiments can be utilized and that the present application can be embodied in other specific forms without departing from the spirit and essential characteristics of the present application. The scope of the present application is therefore to be construed in accordance with the appended claims, including all equivalents of the subject matter of the claims.

Claims

1. A method of treating cancer in a subject in need thereof, the method comprising: administering to the subject a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell in which an endogenous CD5 gene has been knocked out.

2. The method of claim 1, wherein the endogenous CD5 gene is knocked out using a CRISPR method.

3. The method of claim 2, wherein the CRISPR method is a CRISPR / Cas9 method.

4. The method of claim 3, wherein the CRISPR / Cas9 method utilizes an sgRNA comprising the nucleotide sequence of SEQ ID NO:

23.

5. The method of any of the preceding claims, wherein the antigen binding domain of the CAR is capable of binding an antigen selected from the group consisting of CD5, CD19, CD2, CD7, tumor specific antigen (TSA), tumor associated antigen (TAA), a glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, muthsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin, telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, HPV antigens E6, HPV antigens E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alphafetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

6. The method of any one of the preceding claims, wherein the antigen binding domain of the CAR comprises a complementarity determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36, 43-48, 53-58, 65-70, 83-88, and 95-100.

7. The method of any one of the preceding claims, wherein the antigen binding domain of the CAR comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 41, 51, 63, 75, 81, and 93.

8. The method of any one of the preceding claims, wherein the antigen binding domain of the CAR comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 42, 52, 64, 76, 82, and 94.

9. The method of any one of the preceding claims, wherein the antigen binding domain of the CAR comprises an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79, 80, 91, and 92.

10. The method of any one of the preceding claims, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 37, 38, 49, 59, 60, 71, 72, 77, 78, 89, and 90.

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