Use of CD2 / 5 / 7 knockout anti-CD2 / 5 / 7 chimeric antigen receptor T cells against T cell lymphoma and leukemia

Abstract

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 targeting CD2, CD5, or CD7 and modified cells in which CD2, CD5, or CD7 has been knocked out.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 782,131, filed on December 19, 2018, which is incorporated herein by reference in its entirety. Background of the Invention

[0003] T - cell lymphomas and leukemias are aggressive tumors that originate 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 there are few available treatment methods. Chimeric antigen receptor T - cell (CAR T - cell) therapy has proven effective against B - cell tumors, but successfully expanding 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.

[0004] 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 of the Invention

[0005] 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.

[0006] In one aspect, the present invention 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 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 the endogenous CD5 gene has been knocked out.

[0007] In another aspect, the present invention 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 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 the endogenous CD2 gene has been knocked out.

[0008] In yet another aspect, the present invention 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 in which the endogenous CD5 gene has been knocked out.

[0009] In yet another aspect, the present invention includes a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a first modified cell comprising a CAR, wherein the CAR comprises an antigen-binding domain capable of binding CD7, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell in which the endogenous CD7 gene has been knocked out.

[0010] Another aspect of the present invention 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.

[0011] Yet another aspect of the present invention 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.

[0012] Another aspect of the present invention includes a vector comprising any of the nucleic acids disclosed herein.

[0013] In another aspect, the present invention includes a cell comprising any of the nucleic acids disclosed herein or any of the vectors disclosed herein.

[0014] In yet another aspect, the present invention 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 in which the endogenous CD2 gene has been knocked out.

[0015] In yet another aspect, the present invention 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 in which the endogenous CD5 gene has been knocked out.

[0016] In another aspect, the present invention 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 in which the endogenous CD7 gene has been knocked out.

[0017] Another aspect of the present invention 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 in which the endogenous CD5 gene has been knocked out.

[0018] In various embodiments of the above aspects or any other aspects of the invention described herein, an endogenous gene is knocked out using a CRISPR method. In certain embodiments, the CRISPR method is the 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 SEQ ID NO:22 to 24.

[0019] 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), glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, muthsp70-2, M-CSF, prostate enzyme, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin, telomerase, prostate cancer 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 antigen E6, HPV antigen E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\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.

[0020] In certain embodiments, the antigen-binding domain of the CAR comprises complementarity-determining regions (CDRs) that comprise amino acid sequences selected from 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 complementarity-determining regions (CDRs) that comprise amino acid sequences selected from SEQ ID NOs: 31-36, 43-48, 53-58, and 65-70. In certain embodiments, the antigen-binding domain of the CAR comprises complementarity-determining regions (CDRs) that comprise amino acid sequences selected from SEQ ID NOs: 83-88 and 95-100.

[0021] In certain embodiments, the antigen-binding domain of the CAR comprises a heavy-chain variable region that comprises an amino acid sequence selected from 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 that comprises an amino acid sequence selected from SEQ ID NOs: 30, 42, 52, 64, 76, 82, and 94.

[0022] In certain embodiments, the antigen-binding domain of the CAR comprises a heavy-chain variable region that comprises an amino acid sequence selected from SEQ ID NOs: 29, 41, 51, and 63. In certain embodiments, the antigen-binding domain of the CAR comprises a light-chain variable region that comprises an amino acid sequence selected from SEQ ID NOs: 30, 42, 52, and 64.

[0023] In certain embodiments, the antigen-binding domain of the CAR comprises a heavy-chain variable region that comprises an amino acid sequence selected from SEQ ID NOs: 75, 81, and 93. In certain embodiments, the antigen-binding domain of the CAR comprises a light-chain variable region that comprises an amino acid sequence selected from SEQ ID NOs: 76, 82, and 94.

[0024] In certain embodiments, the antigen-binding domain of the CAR comprises a scFv that comprises an amino acid sequence selected from 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 that comprises an amino acid sequence selected from SEQ ID NOs: 27, 28, 39, 40, 50, 61, and 62. In certain embodiments, the antigen-binding domain of the CAR comprises a scFv that comprises an amino acid sequence selected from SEQ ID NOs: 73, 74, 79, 80, 91, and 92.

[0025] In certain embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 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 SEQ ID NO: 25, 26, 37, 38, 49, 59, and 60. In certain embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 71, 72, 77, 78, 89, and 90.

[0026] In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from SEQ ID NO: 1 to 13. In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from SEQ ID NO: 1 to 7. In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from SEQ ID NO: 8 to 13.

[0027] In certain embodiments, the CAR further comprises a suicide gene. In certain embodiments, the suicide gene is iCaspase9.

[0028] In certain embodiments, the first and / or second modified cell is a T cell.

[0029] In certain embodiments, the cancer includes T cell lymphoma or T cell leukemia. In certain embodiments, the cancer is selected from acute myeloid leukemia (AML), T cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0030] In certain embodiments, the composition comprises a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustrating the invention, exemplary embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the precise arrangements and instrumentalities shown in the embodiments of the drawings.

[0032] Figure 1 is a schematic diagram illustrating the current problems of CART therapy for T cell tumors.

[0033] Figure 2 is a schematic diagram illustrating the development of an innovative strategy, wherein: i. gene editing is used to remove tumor targets from normal T cells and thus avoid fratricide during 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 anti-T cell tumor CART to provide T cell immunity that is not affected by CART killing.

[0034] Figure 3 It is a schematic diagram of a new method for targeting T cell lymphoma without causing T cell toxicity. Using a two-pronged immunotherapy, which includes anti-T-NHL (or T-ALL) CART (one of CART2 / 5 / 7, such as CART2) and CD2 / 5 / 7 knockout normal T cells. CART will destroy tumor cells but also kill normal T cells. The infusion of CD2 / 5 / 7 (or other tumor targets) KO normal T cells will provide CART-resistant T cell immunity until CART cells are depleted.

[0035] Figure 4 Examples of anti-CD2 and anti-CD5 CAR constructs used herein. All constructs have a lentiviral pTRPE4-1BB CD3ζ backbone.

[0036] Figure 5 Examples of CART transduction efficiency in T cells. Six different CAR5 constructs were generated using single-chain variable fragments (scFvs) with high (#17), medium (#34), and low (#9) affinities. T cells were activated with anti-CD3 / CD28 beads (Dynabeads), and 24 hours later, a lentiviral vector was added at an MOI of 3. Dynabeads were removed on day 6. CART cells were frozen when the average volume was below 350 fl. CAR expression (goat anti-mouse Fab antibody) was tested on day 6.

[0037] Figure 6 Examples of the CD5 (or CD2, or CD7) KO manufacturing process and CRISPR-Cas9 KO efficiency.

[0038] Figure 7 Examples of the amplification curves of several CART groups. Without CD2 KO, CART2 cells do not expand. With KO, CART2 and CART5 reach approximately 5-8 fold population doublings.

[0039] Figure 8 Examples of the following finding: In the absence of CRISPR-Cas9 KO of CD5, the mean fluorescence intensity (MFI) of CD5 in CART5 is 10-fold lower compared to control T cells, while another pan T cell marker (such as CD2) does not change.

[0040] Figure 9 Examples of CART2 and CART5 amplification curves. T cell concentration was measured using a Coulter Counter.

[0041] Figure 10Illustrates the results from experiments where six different CAR2 constructs were co-cultured with luciferase+Jurkat cells (a T-cell leukemia cell line) and challenged in vitro. At 24 hours, total killing was measured as the relative reduction in luminescence. Only C3029, C3030, and C3043 showed anti-tumor effects.

[0042] Figure 11 Illustrates the results from experiments where six different CAR5 constructs were co-cultured with luciferase+Jurkat cells (a T-cell leukemia cell line) and challenged in vitro. At 24 hours, total killing was measured as the relative reduction in luminescence. All CAR5 constructs showed similar anti-tumor effects.

[0043] Figure 12 Illustrates the in vivo efficacy of CART2 and CART5. NSG mice were implanted with luciferase+Jurkat cells, and the mice were randomly administered control T cells or CART2 or CART5 (1x10 6 ) on day 7. Mice were imaged weekly using an IVIS Xenogen Spectrum and analyzed using Livinglmage software. CART2 C3043 and CART5 C3054 were the most effective.

[0044] Figure 13 Illustrates the results from experiments where Jurkat cells were transduced with different CAR5 constructs (target epitopes and affinities shown on the left) and a GFP-NFAT reporter gene, and then co-cultured with CD5+ tumor cells (or control) for 24 hours. Leading CART5 (C3054) showed increased NFAT activation.

[0045] Figure 14 Illustrates the results from experiments where Jurkat cells were transduced with different CAR2 constructs and a GFP-NFAT reporter gene, and then co-cultured with CD2+ tumor cells (or control) for 24 hours. Leading CART2 (C3043) showed increased NFAT activation.

[0046] Figure 15 Illustrates the CART2 and CART5 activities against cutaneous T-cell lymphoma. Results of a 24-hour killing assay are shown. CART2 cells are active against primary Sezary cells (leukemic cutaneous T-cell lymphoma) and the HH Sezary cell line. CART5 is also active against HH cells.

[0047] Figure 16Illustrates the following finding: CART2 and CART5 can recognize normal T cells (autologous on top and allogeneic on the bottom) and kill them.

[0048] Figure 17 Illustrates the following finding: Removal of the CAR target 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.

[0049] Figure 18 Illustrates the following finding: CMV-specific T cells are present in CD2KO and CD5KO 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 PP65NLVPMVATV dextramer (SEQ ID NO:101); ICS 4 hours after exposure to CETF peptide. After secondary culture with CMV-peptide pulsed APCs).

[0050] Figure 19 Illustrates the development of bispecific CAR T cells. Two lentiviral constructs were generated, which included CAR5 (C3054) and CAR2 (C3043) linked by a P2A sequence. Gene expression was driven by the EF1α promoter. The CAR5 construct has a 4-1BB co-stimulatory domain and a CD3ζ signaling domain.

[0051] Figure 20A - 20B Illustrates bispecific KO CART cells. Efficient knockout of CD2 and CD5 in normal T cells was demonstrated by flow cytometry.

[0052] Figure 21 Illustrates the following finding: In vivo, CD5 KO CART5 is more effective than CD5+ CART5. CD5 KO increases the anti-tumor efficacy of CART5. In a xenograft model of Jurkat T-ALL using NSG mice, compared to WT CART5, CD5KO CART5 (2x10 6 cells / mouse) resulted in complete long-term complete responses and longer survival.

[0053] Figure 22Illustrates the following findings: In vivo, CD5 KO CART19 is more effective than CD5+ CART19. CD5 KO increases the anti-tumor efficacy of CART19. In the NALM6 B-ALL xenograft model, CD5 KO CART19 has significantly higher tumor control compared to WT CART19.

[0054] Figure 23A - 23B Illustrates the following findings: CART5 and CART2 can target 20% of AML. Figure 23A Illustrates CD2 expression in AML. Figure 23B Illustrates the results from a 24-hour killing assay. CART2 cells were co-cultured with CD2+ AML cells and showed significant killing at 24 hours.

[0055] Figure 24 Illustrates the following findings: CART5 can target 100% of CLL and MCL. Results from cytotoxicity assays show that CART5 cells can recognize and kill CD5+ MCL cell lines (Jeko-1 and Mino).

[0056] Figure 25 Illustrates the development of sgRNAs for knocking out CD7 in T cells (top) and the generation of six CAR constructs against CD7.

[0057] Figure 26 Illustrates the Casp9-CAR5 lentiviral construct used herein. Two lentiviral constructs were generated, including CAR5 (C3054), a P2A sequence, and then the iCaspase9 suicide gene (iC9) or iC9-P2A-C3054. Gene expression is driven by the EF1α promoter. The CAR5 construct has a 4-1BB co-stimulatory domain and a CD3ζ signaling domain. Detailed Description

[0058] Definition

[0059] 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 invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice of testing the invention, the preferred materials and methods are described herein. In describing and claiming the invention, the following terms will be used.

[0060] It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0061] As used herein, the articles "a" and "an" refer to one or more than one (i.e., to at least one) of the grammatical objects of the article. By way of example, "a component" means one component or more than one component.

[0062] As used herein, "about" when referring to a measurable value such as an amount, a time period, etc., means encompassing variations of ±20% or ±10% from the specific value, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, provided such variations are suitable for practicing the disclosed methods.

[0063] "Activated", as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" etc. refers to a T cell that has undergone cell division.

[0064] The term "antibody", as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. The antibody can be a complete immunoglobulin derived from a natural source or a recombinant source and can be an immunoreactive portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention 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).

[0065] The term "antibody fragment" refers to a portion of a complete antibody and refers to the antigen-determining variable region of a complete 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 multispecific antibodies.

[0066] "Antibody heavy chain", as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

[0067] "Antibody light chain", as used herein, refers to the smaller of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation, and the κ and λ light chains refer to the two major antibody light chain isotypes.

[0068] The term "synthetic antibody" as used herein means an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a phage as described herein. The term should also be construed to mean an antibody that has been produced by the synthesis of a DNA molecule that encodes the antibody and that expresses the antibody protein or specifies the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0069] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response, which may involve antibody production, or activation of specific immunocompetent cells, or both. One of ordinary skill in the art will understand that any macromolecule - virtually including all proteins or peptides - can be used as an antigen. In addition, an antigen can be derived from recombinant or genomic DNA. One of ordinary skill in the art will understand that any DNA - which includes a nucleotide sequence or a partial nucleotide sequence that encodes a protein that elicits an immune response and thus encodes an "antigen" as the term is used herein. In addition, one of ordinary skill in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in different combinations to elicit a desired immune response. In addition, one of ordinary skill in the art will understand 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 biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0070] As used herein, the term "autologous" means with respect to any substance derived from the same individual that is subsequently reintroduced into that individual.

[0071] "Allogeneic" refers to any substance derived from different animals of the same species.

[0072] "Xenogeneic" refers to any substance derived from animals of different species.

[0073] The term "chimeric antigen receptor" or "CAR", as used herein, refers to an artificial T cell receptor that is engineered to be expressed on an immune effector cell and specifically bind an antigen. CARs can be used as adoptive cell transfer therapies. 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, such as a B cell surface receptor. A CAR can also include an intracellular activation domain, a transmembrane domain, and an extracellular domain that includes a tumor-associated antigen binding region. In some aspects, the CAR includes an extracellular domain that includes an anti-B cell binding domain fused to a CD3-ζ transmembrane domain and intracellular domain.

[0074] The term "cleavage" 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 phosphodiester bonds. Both single-strand cleavage and double-strand cleavage are possible. Double-strand cleavage can occur as a result of two distinct single-strand cleavage events. DNA cleavage can result in the production of blunt ends or staggered ends. In certain embodiments, a fusion polypeptide can be used to target double-stranded DNA for cleavage.

[0075] As used herein, the term "conservative sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an 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 having 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-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 the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the ability of the altered antibody to bind an antigen can be tested using the functional assays described herein.

[0076] As used herein, the term "costimulatory ligand" includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to cognate costimulatory molecules on T cells, thereby providing a signal that mediates a T cell response in addition to the primary signal provided, for example, by binding of the TCR / CD3 complex to peptide-loaded MHC molecules, the T cell response 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 β receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also encompass, in particular, 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 ligands that specifically bind to CD83.

[0077] "Costimulatory molecule" refers to the cognate binding partner on T cells that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response through 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.

[0078] As used herein, the term "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 engagement, results in T cell proliferation and / or upregulation or downregulation of key molecules.

[0079] "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 compared to when it is not in the disorder. Left untreated, a disorder does not necessarily result in a further decrease in the state of health of the animal.

[0080] As used herein, the term "downregulation" refers to a decrease or elimination of the gene expression of one or more genes.

[0081] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, preparation, substance or composition that is effective to achieve a specific biological result or provide a therapeutic or prophylactic benefit as described herein. Such results can include, but are not limited to, anti-tumor activity determined by any suitable means in the art.

[0082] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, where the polymers and macromolecules have either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the nucleotide sequence that is identical to the mRNA sequence and typically provided in the coding strand of the sequence listing, and the non-coding strand that serves as the template for transcribing the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0083] As used herein, "endogenous" refers to any substance that is from or produced within an organism, cell, tissue or system.

[0084] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue or system.

[0085] As used herein, the term "amplification" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, the in vitro amplified T cells increase in number relative to the number initially present in the culture. In another embodiment, the in vitro amplified T cells increase in number relative to other cell types in the culture. The term "in vitro", 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 culture dish, test tube or bioreactor).

[0086] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0087] "Expression vector" refers to a vector containing a recombinant polynucleotide, the recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) incorporating the recombinant polynucleotide, and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0088] As used herein, "homologous" refers to subunit sequence identity between two polymeric molecules (e.g., between two nucleic acid molecules (such as two DNA molecules or two RNA molecules), or between two polypeptide molecules). When the subunit positions in both of the two molecules are occupied by the same monomeric subunit; for example, if the position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two sequences (e.g., five positions in a polymer of ten amino acids) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, the two sequences are 90% homologous.

[0089] A "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as the Fv, Fab, Fab’, F(ab’)2 or other antigen-binding sequences of an antibody) that contains minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the complementarity-determining regions (CDRs) from the recipient are replaced by residues of CDRs from a non-human species (donor antibody), such as a mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, the Fv framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. Additionally, a humanized antibody may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. Generally, a humanized antibody will contain substantially all of at least one and usually two variable domains, in which all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are those of the human immunoglobulin sequence. Preferably, a humanized antibody will also contain at least a portion of the immunoglobulin constant region (Fc), usually the constant region 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.

[0090] "Fully human" refers to immunoglobulins, such as antibodies, in which the entire molecule is of human origin or consists of the same amino acid sequence as a human form of the antibody.

[0091] As used herein, "identity" refers to subunit sequence identity between two polymer molecules, particularly between two amino acid molecules (such as between two polypeptide molecules). When two amino acid sequences have the same residue at the same position; for example, if the position in each of two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue 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 in two sequences (e.g., five positions in a polymer that is ten amino acids in length) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) match or are identical, the two amino acid sequences are 90% identical.

[0092] As used herein, the term "immunoglobulin" or "Ig" 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 receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital 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 effective immunoglobulin in agglutination reactions, complement fixation, and other antibody responses, and is important in defending against bacteria and viruses. IgD is an immunoglobulin that does not have known antibody functions but can function as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing the release of mediators from mast cells and basophils after exposure to an allergen.

[0093] As used herein, the term "immune response" is defined as the cellular response to an antigen that occurs when lymphocytes recognize the antigen molecule as foreign and induce antibody formation and / or activate lymphocytes to eliminate the antigen.

[0094] When referring to an "immunologically effective amount" or "therapeutic amount", the exact amount of the compositions of the present invention to be administered can be determined by a physician or researcher taking into account the age, weight, tumor size, degree of infection or metastasis, and individual differences in the condition of the patient (subject).

[0095] As used herein, "instructions" include publications, records, charts, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The instructions for the kits of the present invention can, for example, be attached to the container containing the nucleic acids, peptides, and / or compositions of the present invention, or be shipped together with the container containing the nucleic acids, peptides, and / or compositions. Optionally, the instructions can be shipped separately from the container, with the intention that the instructions and the compounds be used in combination by the recipient.

[0096] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that naturally occurs in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the coexisting substances in its natural state is "isolated". The isolated nucleic acid or protein can exist in a substantially purified form, or, for example, can exist in a non-natural environment such as a host cell.

[0097] As used herein, the term "knockdown" refers to a decrease in the gene expression of one or more genes.

[0098] As used herein, the term "knockout" refers to the elimination of the gene expression of one or more genes.

[0099] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Among retroviruses, lentiviruses are the only ones capable of infecting non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, so they are one of the most effective methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide tools for achieving significant levels of in vivo gene transfer.

[0100] As used herein, the term "limited toxicity" means that the peptides, polynucleotides, cells, and / or antibodies of the present invention exhibit a lack of substantially negative biological effects, anti-tumor effects, or substantially negative physiological symptoms on healthy cells, non-tumor cells, non-lesion cells, non-target cells, or populations of these cells, either in vitro or in vivo.

[0101] As used herein, the term "modified" refers to an altered state or structure of the molecules or cells of the present invention. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by introducing nucleic acids.

[0102] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of response in a subject as compared to the level of response in a subject lacking treatment or a compound, and / or as compared to the level of response in an otherwise identical but untreated subject. The term encompasses disrupting and / or affecting natural signals or responses, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0103] In the context of the present invention, the following abbreviations are used for the nucleic acid bases that commonly occur. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0104] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that the nucleotide sequence encoding the protein may include intron(s) in some versions.

[0105] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. 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 two protein-coding regions must be joined in the same reading frame.

[0106] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate an abnormal level of tumor antigen expression in cells of a solid tumor within a specific tissue or organ of a patient, such as a diseased area, relative to the expression level in normal cells from the tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of a tumor antigen can be determined by standard assays known in the art.

[0107] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection, or infusion techniques.

[0108] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acids and polynucleotides are used interchangeably herein. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides 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 recombinant libraries or cell genomes, using conventional cloning techniques and PCR TM and so on to clone nucleic acid sequences, and synthetic means.

[0109] 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 in the sequence that can comprise a protein or peptide. A polypeptide includes any peptide or protein that contains two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, for example; and longer chains, which are commonly referred to in the art as proteins, which have many 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. Polypeptides include natural peptides, recombinant peptides, synthetic peptides or combinations thereof.

[0110] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery that is required to initiate the specific transcription of a polynucleotide sequence.

[0111] "Signal transduction pathway" refers to the biochemical relationship between various signal transduction molecules that function 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 capable of receiving and transmitting signals across the plasma membrane of a cell.

[0112] As used herein, the term "specifically binds" with respect to an antibody means an antibody that recognizes a specific antigen, but substantially does not recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species may also bind antigens from one or more species. However, this cross-species reactivity per se does not change the class of the antibody by specificity. In another example, an antibody that specifically binds an antigen may also bind different allelic forms of the antigen. However, this cross-reactivity per se does not change the class of the antibody by specificity. In some instances, the terms "specific binding" or "binds specifically" may 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 specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody generally recognizes and binds a specific protein structure rather than recognizing and binding proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0113] By the term "stimulate" is meant the primary response induced by binding a stimulatory molecule (e.g., the TCR / CD3 complex) to its associated ligand, thereby mediating signal transduction events - such as but not limited to signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β and / or reorganization of the cytoskeletal structure, etc.

[0114] "Stimulatory molecule", as used herein, means a molecule on a T cell that specifically binds to an associated stimulatory ligand present on an antigen-presenting cell.

[0115] As used herein, "stimulatory ligand" means such a ligand that, when present on an antigen-presenting cell (e.g., an aAPC, dendritic cell, B-cell, etc.), can specifically bind to an associated binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating the primary response of the T cell, which includes but is not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, in particular, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0116] 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 non-human mammal. Non-human mammals include, for example, domestic and pet animals such as sheep, cattle, pigs, dogs, cats, and murine mammals. Preferably, the subject is a human.

[0117] 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 cell types that are normally associated with it in its native state. In some examples, a substantially purified cell population refers to a homogeneous cell population. In other examples, the term simply refers to a cell that has been separated from the cells that are normally associated with it in its native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0118] A "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient to effect binding.

[0119] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins that is involved in the activation of T cells in response to antigen presentation. The TCR is responsible for recognizing antigens that are bound to major histocompatibility complex molecules. The TCR consists of a heterodimer of alpha (α) and beta (β) chains, but in some cells the TCR consists of gamma and delta (γ / δ) chains. The TCR can exist in α / 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 the TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0120] As used herein, the term "treated" means treatment and / or prophylaxis. A therapeutic effect is obtained by inhibiting, alleviating, or eliminating a disease state.

[0121] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the 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.

[0122] As used herein, the term "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0123] As used herein, the phrase "under transcriptional control" or "operatively linked" means that a promoter is in the correct position and orientation relative to a polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0124] "Vector" refers to a substance composition that includes isolated nucleic acids and can be used to deliver the isolated nucleic acids into cells. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-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.

[0125] Ranges: Throughout this disclosure, aspects of the invention may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0126] Description

[0127] This disclosure describes three chimeric antigen receptors (CARs) that target T cell tumors and methods for preventing healthy T cells from killing each other. T cell lymphomas and leukemias are aggressive tumors that originate 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 there are few available treatments. CAR T cell therapy has proven effective against B cell tumors, but until now, extending the success of chimeric antigen receptor (CAR) T cells to T cell malignancies has been problematic 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 potential lethal immunosuppression resulting from the elimination of the T cell compartment.

[0128] In certain embodiments, the CAR targets the T cell antigens CD2, CD5, and CD7. In certain embodiments, CRISPR-Cas knocks out the CD2, CD5, or CD7 targets in healthy T cells, preventing the killing of healthy T cells during manufacturing and subsequent CAR T cell therapy.

[0129] Therapeutic method

[0130] The present invention includes methods of treating T cell lymphoma or T cell leukemia in a subject in need thereof. In another aspect, the present invention includes methods of preventing autologous killing of CAR T cells in a subject in need thereof.

[0131] In certain embodiments, 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 targeting CD2, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell in which the endogenous CD2 gene has been knocked out.

[0132] In certain embodiments, the method includes 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 the endogenous CD5 gene has been knocked out.

[0133] In certain embodiments, the method includes 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 the endogenous CD7 gene has been knocked out.

[0134] In certain embodiments, 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 the endogenous CD5 gene has been knocked out.

[0135] In certain embodiments, the method includes 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 the endogenous CD5 gene in the cell has been knocked out.

[0136] In various embodiments of the methods disclosed herein, any of the CARs disclosed herein can be administered to the subject. The CAR is specific for any tumor-associated antigen (TAA) or tumor-specific antigen (TSA) known to those of ordinary skill in the art.

[0137] In certain embodiments, the CAR comprises complementarity determining regions (CDRs) that comprise amino acid sequences selected from 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 that includes a heavy chain variable region comprising an amino acid sequence selected from 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 SEQ ID NOs: 30, 42, 52, 64, 76, 82, and 94. In certain embodiments, the CAR comprises a scFv that comprises an amino acid sequence selected from SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79, 80, 91, and 92.

[0138] In certain embodiments, an object is administered a CAR that comprises a nucleic acid sequence encoded by any one of SEQ ID NOs: 1 to 13. In certain embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NOs: 25, 26, 37, 38, 49, 59, 60, 71, 72, 77, 78, 89, and 90.

[0139] In certain embodiments, the first and / or second modified cell is a T cell. In certain embodiments, the cancer includes T cell lymphoma or T cell leukemia. Cancer types that can be treated with the compositions and methods of the invention include, but are not limited to, non-Hodgkin's lymphoma and its subtypes, including peripheral T-cell lymphoma (PTCL), angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL, ALK-), natural killer / T cell lymphoma (NKTCL), adult T cell leukemia / lymphoma (ATLL), ALCL ALK+, enteropathy-type T cells, hepatosplenic T cells, subcutaneous panniculitis-like, and unclassified PTCL.

[0140] In certain embodiments, endogenous genes (such as CD2, CD5, and CD7) are knocked out using the CRISPR / Cas9 method. In certain embodiments, the CRISPR / Cas9 method utilizes an sgRNA targeting CD2, CD5, and / or CD7. In certain embodiments, the sgRNA comprises a nucleotide sequence selected from SEQ ID NOs: 22 to 24.

[0141] In certain embodiments, the CAR of the present invention further comprises a suicide gene. A 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 the fusion of human caspase 9 with a modified human FK-binding protein, allowing conditional dimerization using a small molecule drug (e.g., AP1903). When exposed to the synthetic dimerizing drug, iCaspase9 becomes activated and causes rapid apoptosis of cells expressing the construct (e.g., CAR T cells) (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 after expression, it converts the non-toxic prodrug GCV into GCV-triphosphate, leading to cell death by halting DNA replication.

[0142] Composition

[0143] One aspect of the present invention 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 in which the endogenous CD2 gene has been knocked out.

[0144] Another aspect of the present invention 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 in which the endogenous CD5 gene has been knocked out.

[0145] Yet another aspect of the present invention 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 in which the endogenous CD7 gene has been knocked out.

[0146] In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from SEQ ID NOs: 1 to 13. In certain embodiments, the endogenous gene has been knocked out using the CRISPR / Cas system. In certain embodiments, the CRISPR / Cas9 system comprises a gRNA, and the gRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 22 to 24.

[0147] The present invention further includes the composition of the present invention and a pharmaceutically acceptable carrier.

[0148] Chimeric antigen receptor (CAR)

[0149] The present invention provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the present invention includes a CAR comprising an antigen-binding domain capable of binding CD2, a transmembrane domain, and an intracellular domain.

[0150] Antigen - binding domain

[0151] In one embodiment, the CAR of the present invention comprises a target-specific binding element, also referred to as an antigen-binding domain. The selection of the antigen-binding domain depends on the type and number of ligands that define the surface of the target cell. For example, an antigen-binding domain can be selected to recognize a ligand that serves as a cell surface marker on target cells associated with a particular disease state (e.g., T cell lymphoma or leukemia).

[0152] In one embodiment, the CAR of the present invention can be engineered to target tumor antigens. The antigens discussed herein are included only as examples. The list is not intended to be exclusive, and further examples will be apparent to those skilled in the art. Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding domain of the present invention will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostasin, PSMA, Her2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0153] The tumor antigens of this type mentioned in the present invention 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. Antigens associated with TAAs are not unique to tumor cells and, on the contrary, are also expressed on normal cells in conditions that do not induce an immune tolerance state to the antigen. Antigen expression on tumors can occur in conditions that enable the immune system 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 normally exist at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0154] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-l / MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens generated by chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA125, 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-related protein, TAAL6, TAG72, TLP, and TPS.

[0155] Depending on the desired antigen to be targeted, the CARs of the present invention can be engineered to include an appropriate antigen-binding domain that is 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 CARs of the present invention.

[0156] 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.

[0157] In some embodiments, the antigen-binding domain in the CARs of the present invention is an anti-CD2 scFV. In some embodiments, the antigen-binding domain in the CARs of the present invention is an anti-CD5 scFV. In some embodiments, the antigen-binding domain in the CARs of the present invention 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.

[0158] In certain embodiments, the antigen-binding domain comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprising three heavy-chain complementarity-determining regions (HCDRs), and the light-chain variable region comprising three light-chain complementarity-determining regions (LCDRs).

[0159] In certain embodiments, the present invention includes a CAR that includes an antigen-binding domain capable of binding CD2, wherein the antigen-binding domain comprises complementarity-determining regions (CDRs) that include the amino acid sequence of any one of SEQ ID NO: 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.

[0160] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding CD2, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 31, HCDR2 comprises the amino acid sequence of SEQ ID NO: 32, HCDR3 comprises the amino acid sequence of SEQ ID NO: 33, LCDR1 comprises the amino acid sequence of SEQ ID NO: 34, LCDR2 comprises the amino acid sequence of SEQ ID NO: 35, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 36.

[0161] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding CD2, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 43, HCDR2 comprises the amino acid sequence of SEQ ID NO: 44, HCDR3 comprises the amino acid sequence of SEQ ID NO: 45, LCDR1 comprises the amino acid sequence of SEQ ID NO: 46, LCDR2 comprises the amino acid sequence of SEQ ID NO: 47, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 48.

[0162] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding CD2, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 65, HCDR2 comprises the amino acid sequence of SEQ ID NO: 66, HCDR3 comprises the amino acid sequence of SEQ ID NO: 67, LCDR1 comprises the amino acid sequence of SEQ ID NO: 68, LCDR2 comprises the amino acid sequence of SEQ ID NO: 69, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 70.

[0163] In certain embodiments, the CAR comprises an antigen-binding domain 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.

[0164] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding CD2, wherein the antigen-binding domain is a scFv, and the scFv comprises the amino acid sequence set forth in any one of SEQ ID NO: 27, 28, 39, 40, 50, 61 or 62.

[0165] In certain embodiments, the present invention includes a CAR, the CAR comprising an antigen-binding domain capable of binding to CD5, wherein the antigen-binding domain comprises complementarity-determining regions (CDRs), the complementarity-determining regions comprising the amino acid sequence of any one of SEQ ID NO: 83, 84, 85, 86, 87, 88, 95, 96, 97, 98, 99 or 100.

[0166] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to 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.

[0167] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to 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.

[0168] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to 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.

[0169] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to CD5, wherein the antigen-binding domain is a scFv comprising the amino acid sequence of any one of SEQ ID NO: 73, 74, 79, 80, 91 or 92.

[0170] Permissible variations in 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 NO: 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.

[0171] Transmembrane domain

[0172] Regarding the transmembrane domain, the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is native to 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 the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

[0173] The transmembrane domain can be derived from a natural source or a synthetic source. When the source is natural, the domain can be derived from any membrane-binding or transmembrane protein. Transmembrane regions for particular uses in the present invention can be derived from the α, β, or ζ chains of the T-cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., at least including the above transmembrane region(s)). Optionally, the transmembrane domain can be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR. The glycine-serine doublet provides a particularly suitable linker.

[0174] In one embodiment, the transmembrane domain in the CAR of the present invention is the 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 the 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.

[0175] In some cases, the transmembrane domain of the CAR of the present invention comprises the CD8α 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 the 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.

[0176] 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 refers to any oligopeptide or polypeptide that serves to link the transmembrane domain to an extracellular domain or a cytoplasmic domain in a 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.

[0177] Intracellular domain

[0178] The intracellular domain or additional cytoplasmic domain of the CAR of the present invention is responsible for activating at least one normal effector function of the immune cell in which the CAR has been placed. The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity including cytokine secretion. Thus, the term "intracellular domain" refers to the protein portion that transduces effector function signals and directs the cell to perform its specialized function. Although the entire intracellular domain can generally 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 replace the full chain as long as it transduces effector function signals. Thus, the term intracellular domain is intended to include any truncated portion of the intracellular domain that is sufficient to transduce effector function signals.

[0179] Preferred examples of intracellular domains for use in the CAR of the present invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that cooperate to initiate signal transduction upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0180] It is known that the signals generated by the TCR alone are insufficient to fully activate T cells, and secondary or co-stimulatory signals are also required. Thus, T cell activation can be considered to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0181] The primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0182] Examples of ITAMs containing primary cytoplasmic signaling sequences having particular utility in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Particularly preferably, the cytoplasmic signaling molecule in the CAR of the present invention includes a cytoplasmic signaling sequence derived from CD3ζ.

[0183] In a preferred embodiment, the intracellular domain of the CAR can be designed to itself contain the CD3-ζ signaling domain, or can be combined with any other desired intracellular domain(s) useful in the context of the CAR of the present invention. For example, the intracellular domain of the CAR can include a CD3ζ chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to the part of the CAR that contains the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for an effective response of lymphocytes to an antigen, rather than antigen receptors or their ligands. 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 ligands that specifically bind to CD83, and the like. Thus, although the present invention mainly takes 4-1BB as an example of the co-stimulatory signaling element, other co-stimulatory elements are also within the scope of the present invention.

[0184] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be connected to each other randomly or in a defined order. Optionally, short oligopeptide or polypeptide linkers, preferably having a length between 2 and 10 amino acids, can form the connection. The glycine-serine doublet provides a particularly suitable linker.

[0185] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domains of CD28 and 4-1BB.

[0186] In one embodiment, the intracellular domain in the CAR of the present invention is designed to include the signaling domain of 4-IBB and the signaling domain of CD3-ζ, 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-ζ comprises the nucleic acid sequence set forth in SEQ ID NO:19.

[0187] In one embodiment, the intracellular domain in the CAR of the present invention is designed to include the signaling domain of 4-IBB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-IBB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20, and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:21.

[0188] In one embodiment, the intracellular domain in the CAR of the present invention is designed to include the signaling domain of 4-IBB and the signaling domain of CD3-ζ, 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-ζ comprises the amino acid sequence set forth in SEQ ID NO:21.

[0189] In one embodiment, the anti-CD2 CAR comprises the amino acid sequence set forth in any one of SEQ ID NO:25, 26, 37, 38, 49, 59 or 60. In one embodiment, the anti-CD2 CAR is encoded by a nucleic acid sequence selected from 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 SEQ ID NOs:8 to 13.

[0190] Permissible variations of the CAR sequence will be known to those skilled 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 sequences set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

[0191] The invention should be construed to include any of the following: a CAR, a nucleic acid encoding the CAR, a vector comprising the nucleic acid encoding the CAR, a cell comprising the CAR, a cell comprising the nucleic acid encoding the CAR, and a cell comprising a vector comprising the nucleic acid encoding the CAR.

[0192] CD2 - MEDI507H2L - 3028 CAR (SEQ ID NO:1)

[0193] ggatccCAAGTCCAACTGGTGCAATCAGGCGCAGAAGTCCAACGACCGGGGGCCAGTGTTAAAGTGTCTTGTAAAGCCTCCGGGTACATTTTTACTGAGTACTATATGTACTGGGTCAGACAGGCCCCAGGGCAAGGTTTGGAACTTGTCGGACGCATAGATCCCGAAGACGGTTCTATAGATTACGTTGAGAAGTTCAAAAAGAAAGTCACACTTACTGCGGACACATCTAGTAGCACCGCATATATGGAACTGAGCAGTCTCACCTCAGACGACACCGCAGTGTACTATTGCGCTCGCGGAAAGTTTAACTATAGGTTCGCGTACTGGGGACAGGGGACACTGGTGACTGTTAGCAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGACGTTGTGATGACGCAAAGTCCCCCGTCACTCCTTGTTACTCTCGGCCAGCCAGCGTCTATCTCTTGCCGGTCAAGCCAGAGCTTGCTCCACTCTAGTGGTAACACGTATTTGAACTGGTTGCTGCAAAGGCCTGGACAATCTCCTCAGCCCCTGATCTATTTGGTTAGCAAACTGGAAAGTGGTGTTCCAGACAGATTTTCAGGGTCTGGATCAGGCACTGATTTCACTCTGAAGATCTCCGGGGTAGAGGCCGAGGACGTGGGAGTCTATTACTGCATGCAGTTTACTCACTATCCTTATACCTTTGGTCAAGGGACGAAACTGGAGATCAAAtccgga

[0194] CD2 - OKT11H2L - 3029 CAR (SEQ ID NO:2)

[0195] ggatccCAAGTTCAGCTTCAGCAACCAGGTGCTGAATTGGTCCGCCCTGGAACTAGCGTTAAACTGTCTTGTAAGGCATCCGGTTATACGTTTACAAGTTATTGGATGCACTGGATTAAGCAAAGGCCCGAACAAGGCCTTGAATGGATTGGGAGAATTGATCCCTACGATAGCGAGACACACTACAATGAAAAATTTAAAGATAAGGCCATCCTCAGCGTAGATAAGAGCAGTTCTACCGCATACATACAGCTCTCAAGCCTGACGTCAGATGACTCAGCCGTTTATTATTGCTCAAGGCGGGACGCTAAATACGACGGCTATGCGCTTGACTACTGGGGACAAGGCACCACTTTGACAGTCTCCAGTggtggcggagggagcggcggtggaggaagcggaggcggaggttccGATATAGTTATGACGCAAGCAGCACCCTCTGTACCTGTGACACCGGGTGAATCCGTTAGTATCTCATGCCGCTCTTCTAAAACCCTCTTGCATTCTAACGGCAATACATATTTGTATTGGTTCCTTCAACGACCAGGACAATCACCGCAAGTGCTTATTTATAGGATGTCTAACTTGGCTAGTGGGGTGCCAAATAGGTTCAGTGGGTCTGGATCTGAGACAACTTTCACGTTGAGAATAAGTAGGGTGGAAGCTGAAGACGTCGGTATATACTACTGTATGCAGCATTTGGAGTACCCTTACACTTTCGGGGGAGGTACTAAGCTCGAAATTAAAtccgga

[0196] CD2 - OKT11L2H - 3030 CAR (SEQ ID NO:3)

[0197] ggatccGATATAGTTATGACGCAAGCAGCACCCTCTGTACCTGTGACACCGGGTGAATCCGTTAGTATCTCATGCCGCTCTTCTAAAACCCTCTTGCATTCTAACGGCAATACATATTTGTATTGGTTCCTTCAACGACCAGGACAATCACCGCAAGTGCTTATTTATAGGATGTCTAACTTGGCTAGTGGGGTGCCAAATAGGTTCAGTGGGTCTGGATCTGAGACAACTTTCACGTTGAGAATAAGTAGGGTGGAAGCTGAAGACGTCGGTATATACTACTGTATGCAGCATTTGGAGTACCCTTACACTTTCGGGGGAGGTACTAAGCTCGAAATTAAAggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAAGTTCAGCTTCAGCAACCAGGTGCTGAATTGGTCCGCCCTGGAACTAGCGTTAAACTGTCTTGTAAGGCATCCGGTTATACGTTTACAAGTTATTGGATGCACTGGATTAAGCAAAGGCCCGAACAAGGCCTTGAATGGATTGGGAGAATTGATCCCTACGATAGCGAGACACACTACAATGAAAAATTTAAAGATAAGGCCATCCTCAGCGTAGATAAGAGCAGTTCTACCGCATACATACAGCTCTCAAGCCTGACGTCAGATGACTCAGCCGTTTATTATTGCTCAAGGCGGGACGCTAAATACGACGGCTATGCGCTTGACTACTGGGGACAAGGCACCACTTTGACAGTCTCCAGTtccgga

[0198] CD2 - T11 - 2 - H2L - 3031 CAR (SEQ ID NO:4)

[0199] ggatccCAAGTTCAATTGCAGCAACCGGGTGCCGAGTTGGTAAGGCCCGGTGCGTCAGTCAAACTTAGTTGTAAAGCTAGTGGGTACACTTTTACTACGTTCTGGATGAATTGGGTGAAGCAACGACCAGGCCAAGGTCTGGAATGGATCGGCATGATTGACCCGTCTGACTCAGAAGCTCATTACAACCAGATGTTCAAGGACAAGGCGACTCTGACTGTTGATAAAAGCTCAAGCACCGCCTACATGCAGCTCAGTAGCCTCACATCCGAGGATTCCGCAGTGTACTATTGCGCGAGGGGACGAGGGTATGATGACGGCGATGCGATGGACTATTGGGGACAGGGGACCAGCGTAACAGTCAGTAGTggtggcggagggagcggcggtggaggaagcggaggcggaggttccGATATAGTTATGACCCAGTCTCCCGCCTCTCTGGCCGTTAGCTTGGGACAACGCGCTACCATCTCTTACCGAGCGTCTAAGTCCGTCAGTACAAGCGGTTATAGTTACATGCACTGGAACCAGCAAAAGCCCGGACAACCTCCGAGACTCCTGATTTATTTGGTCTCTAACCTTGAGTCAGGTGTCCCAGCCAGATTCTCCGGCTCTGGAAGCGGCACTGACTTTACATTGAACATTCACCCCGTGGAGGAGGAAGACGCTGCTACCTACTATTGCATGCAATTCACGCACTATCCCTACACATTCGGGGGGGGCACGAAATTGGAAATCAAAtccgga

[0200] CD2 - TS2 - 18.1.1 - H2L - 3032 CAR (SEQ ID NO:5)

[0201] ggatccGAGGTTCAGCTTGAGGAGAGTGGGGGAGGTTTGGTAATGCCAGGTGGGTCTTTGAAACTCAGTTGCGCGGCGTCAGGCTTCGCATTTTCCTCCTACGATATGTCCTGGGTCAGACAGACACCCGAGAAGCGGCTGGAATGGGTCGCTTACATTTCCGGGGGAGGATTCACGTACTACCCGGATACAGTAAAGGGGAGATTTACTCTGAGCCGGGACAACGCTAAGAATACCCTCTATCTCCAGATGTCCTCTTTGAAGAGTGAAGACACAGCGATGTATTACTGTGCGAGACAAGGGGCCAATTGGGAGCTGGTTTACTGGGGCCAGGGGACGACATTGACGGTTTCTAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGACATTGTAATGACACAATCACCTGCTACACTTAGCGTGACTCCAGGTGATCGGGTATTCCTGAGCTGCCGCGCATCACAAAGTATATCCGACTTCCTGCACTGGTATCAGCAGAAATCTCACGAAAGTCCCAGGCTGCTGATTAAATACGCTTCCCAGAGTATTAGTGGTATCCCCTCACGATTTTCTGGCAGCGGGAGCGGTAGTGACTTCACTCTTTCTATAAACTCCGTCGAGCCAGAAGACGTGGGGGTGTATCTTTGCCAAAATGGACACAATTTTCCACCAACCTTTGGTGGGGGCACCAAACTCGAAATAAAGtccgga

[0202] CD2 - TS2 - 18.1.1 - L2H - 3033 CAR (SEQ ID NO:6)

[0203] ggatccGACATTGTAATGACACAATCACCTGCTACACTTAGCGTGACTCCAGGTGATCGGGTATTCCTGAGCTGCCGCGCATCACAAAGTATATCCGACTTCCTGCACTGGTATCAGCAGAAATCTCACGAAAGTCCCAGGCTGCTGATTAAATACGCTTCCCAGAGTATTAGTGGTATCCCCTCACGATTTTCTGGCAGCGGGAGCGGTAGTGACTTCACTCTTTCTATAAACTCCGTCGAGCCAGAAGACGTGGGGGTGTATCTTTGCCAAAATGGACACAATTTTCCACCAACCTTTGGTGGGGGCACCAAACTCGAAATAAAGggtggcggagggagcggcggtggaggaagcggaggcggaggttccGAGGTTCAGCTTGAGGAGAGTGGGGGAGGTTTGGTAATGCCAGGTGGGTCTTTGAAACTCAGTTGCGCGGCGTCAGGCTTCGCATTTTCCTCCTACGATATGTCCTGGGTCAGACAGACACCCGAGAAGCGGCTGGAATGGGTCGCTTACATTTCCGGGGGAGGATTCACGTACTACCCGGATACAGTAAAGGGGAGATTTACTCTGAGCCGGGACAACGCTAAGAATACCCTCTATCTCCAGATGTCCTCTTTGAAGAGTGAAGACACAGCGATGTATTACTGTGCGAGACAAGGGGCCAATTGGGAGCTGGTTTACTGGGGCCAGGGGACGACATTGACGGTTTCTAGCtccgga

[0204] CD2 - MEDI507L2H - 3043 CAR (SEQ ID NO:7)

[0205] ggatccGACGTTGTGATGACGCAAAGTCCCCCGTCACTCCTTGTTACTCTCGGCCAGCCAGCGTCTATCTCTTGCCGGTCAAGCCAGAGCTTGCTCCACTCTAGTGGTAACACGTATTTGAACTGGTTGCTGCAAAGGCCTGGACAATCTCCTCAGCCCCTGATCTATTTGGTTAGCAAACTGGAAAGTGGTGTTCCAGACAGATTTTCAGGGTCTGGATCAGGCACTGATTTCACTCTGAAGATCTCCGGGGTAGAGGCCGAGGACGTGGGAGTCTATTACTGCATGCAGTTTACTCACTATCCTTATACCTTTGGTCAAGGGACGAAACTGGAGATCAAAggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAAGTCCAACTGGTGCAATCAGGCGCAGAAGTCCAACGACCGGGGGCCAGTGTTAAAGTGTCTTGTAAAGCCTCCGGGTACATTTTTACTGAGTACTATATGTACTGGGTCAGACAGGCCCCAGGGCAAGGTTTGGAACTTGTCGGACGCATAGATCCCGAAGACGGTTCTATAGATTACGTTGAGAAGTTCAAAAAGAAAGTCACACTTACTGCGGACACATCTAGTAGCACCGCATATATGGAACTGAGCAGTCTCACCTCAGACGACACCGCAGTGTACTATTGCGCTCGCGGAAAGTTTAACTATAGGTTCGCGTACTGGGGACAGGGGACACTGGTGACTGTTAGCAGCtccgga

[0206] CD5 - 17L2H - 3045 CAR (SEQ ID NO:8)

[0207] ggatccAACATTGTACTGACGCAAAGCCCCTCATCTTTGTCTGAGTCACTCGGCGGCAAAGTAACCATCACATGCAAGGCCAGTCAAGACATCAATAAATATATTGCTTGGTATCAGTATAAACCCGGCAAGGGGCCGCGACTGCTGATTCACTACACGAGTACCTTGCAACCGGGCATTCCGAGCCGATTTAGTGGCAGTGGCTCAGGTCGCGATTACTCATTCTCAATAAGTAATCTCGAACCGGAAGACATAGCTACTTATTATTGCTTGCAGTACGATAATTTGTGGACCTTCGGGGGTGGTACAAAGTTGGAAATAAAGggtggcggagggagcggcggtggaggaagcggaggcggaggttccGAGGTCCAACTCGTAGAATCAGGTCCCGGATTGGTGCAACCATCCCAGAGCCTCTCTATTACATGCACGGTCTCTGGATTTAGTCTGACCAATTACGATGTGCATTGGGTGCGCCAGTCTCCCGGCAAGGGGTTGGAATGGCTTGGCGTTATATGGAACTACGGAAATACAGACTATAACGCCGCGTTTATCTCTCGGCTGAGTATACGGAAAGACAGTAGTAAATCCCAGGTCTTTTTTACGATGTCATCCCTGCAAACGCCAGATACCGCAATATATTACTGCGCCAGGAACCACGGTGATGGTTATTATAATTGGTACTTCGATGTGTGGGGTACTGGCACTACAGTCACAGTATCTTCAtctaga

[0208] CD5 - 9H2L - 3048 CAR (SEQ ID NO:9)

[0209] ggatccCAG GTC CAG CTG AAA GAA AGC GGT CCA GAG CTG GAA AAA CCC GGT GCGAGC GTC AAA ATA TCA TGT AAA GCA AGC GGG TAT TCA TTC ACC GCG TAC TCT ATG AACTGG GTT AAG CAA AAC AAC GGT ATG TCC TTG GAG TGG ATA GGG TCT ATC GAC CCG TATTAT GGG GAC ACA AAA TAC GCG CAG AAA TTC AAG GGG AAG GCC ACC CTG ACC GTA GATAAA GT AGT TCT ACT GCG TAC TTG CAA CTG AAA AGC CTC ACT TCT GAG GAC TCT GCCGTC TAC TAC TGT GCT CGG CGA ATG ATA ACG ACG GGG GAC TGG TAT TTC GAT GTT TGGGGT ACA GGG ACT ACG GTG ACT GTC AGT AGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAT ATC GTC TTG ACT CAA TCA CCT AGT TCT TTG TCT GCG TCC CTT GGC GACCGA GTC ACC ATA TCT TGC AGA GCG TCA CAG GAC ATT TCA ACG TAC CTC AAC TGG TATCAG CAA AAA CCG GAC GGG ACT GTC AAG CTC TTG ATC TTC TAC ACT TCC AGA CTC CACGCC 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

[0210] CD5 - 9L2H - 3049CAR (SEQ ID NO:10)

[0211] GgatccCAT ATC GTC TTG ACT CAA TCA CCT AGT TCT TTG TCT GCG TCC CTT GGCGAC CGA GTC ACC ATA TCT TGC AGA GCG TCA CAG GAC ATT TCA ACG TAC CTC AAC TGGTAT 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 CTTACA ATC AGC AAT CTT GAG CAA GAA GAC ATA GCC ACG TAT TTC TGC CAG CAA GGT AACTCA CTT CCG TTC ACG TTT GGT AGT GGC ACC AAA CTG GAG ATA AAA ggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAG GTC CAG CTG AAA GAA AGC GGT CCA GAG CTG GAAAAA CCC GGT GCG AGC GTC AAA ATA TCA TGT AAA GCA AGC GGG TAT TCA TTC ACC GCGTAC TCT ATG AAC TGG GTT AAG CAA AAC AAC GGT ATG TCC TTG GAG TGG ATA GGG TCTATC GAC CCG TAT TAT GGG GAC ACA AAA TAC GCG CAG AAA TTC AAG GGG AAG GCC ACCCTG ACC GTA GAT AAA GCT AGT TCT ACT GCG TAC TTG CAA CTG AAA AGC CTC ACT TCTGAG GAC TCT GCC GTC TAC TAC TGT GCT CGG CGA ATG ATA ACG ACG GGG GAC TGG TATTTC GAT GTT TGG GGT ACA GGG ACT ACG GTG ACT GTC AGT AGCtccgga

[0212] CD5 - 34H2L - 3052 CAR (SEQ ID NO:11)

[0213] ggatccGAGGTTAAACTCGTGGAGAGCGGTGCCGAACTCGTCCGAAGTGGTGCTTCCGTTAAACTCAGTTGTGCCGCGTCAGGATTTAACATAAAAGATTACTACATTCACTGGGTCAAACAGCGCCCGGAGCAGGGGCTTGAATGGATCGGGTGGATTGATCCTGAAAACGGGCGCACCGAATATGCTCCCAAGTTCCAGGGCAAAGCTACTATGACCGCTGACACCTCTAGTAACACTGCCTACCTGCAGTTGAGCTCTCTTACGTCTGAGGATACCGCTGTGTACTACTGTAATAACGGAAATTATGTACGACACTATTACTTCGACTACTGGGGGCAGGGCACTACTGTGACTGTATCTAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGATTGGCTCACACAATCCCCTGCAATCCTGAGTGCATCTCCAGGCGAGAAAGTAACTATGACTTGCAGAGCTATAAGCTCTGTGTCCTACATGCACTGGTATCAGCAGAAGCCAGGTTCTTCCCCGAAGCCGTGGATATATGCTACAAGCAATTTGGCATCCGGTGTTCCCGCCCGGTTTAGTGGCTCCGGTTCTGGGACAAGTTACTCCCTCACGATCAGCAGGGTTGAAGCCGAGGACGCTGCCACTTACTATTGCCAACAGTGGTCAAGTAACCCCAGGACTTTCGGGGGAGGAACTAAACTTGAAATCAAAtctaga

[0214] CD5 - 34L2H - 3053 CAR (SEQ ID NO:12)

[0215] GgatccGAT TGG CTC ACA CAA TCC CCT GCA ATC CTG AGT GCA TCT CCA GGC GAGAAA GTA ACT ATG ACT TGC AGA GCT ATA AGC TCT GTG TCC TAC ATG CAC TGG TAT CAGCAG AAG CCA GGT TCT TCC CCG AAG CCG TGG ATA TAT GCT ACA AGC AAT TTG GCA TCCGGT GTT CCC GCC CGG TTT AGT GGC TCC GGT TCT GGG ACA AGT TAC TCC CTC ACG ATCAGC AGG GTT GAA GCC GAG GAC GCT GCC ACT TAC TAT TGC CAA CAG TGG TCA AGT AACCCC AGG ACT TTC GGG GGA GGA ACT AAA CTT GAA ATC AAA

[0216] GgtggcggagggagcggcggtggaggaagcggaggcggaggttccGAG GTT AAACTC GTG GAGAGC GGT GCC GAA CTC GTC CGA AGT GGT GCT TCC GTT AAA CTC AGT TGT GCC GCG TCAGGA TTT AAC ATA AAA GAT TAC TAC ATT CAC TGG GTC AAA CAG CGC CCG GAG CAG GGGCTT GAA TGG ATC GGG TGG ATT GAT CCT GAA AAC GGG CGC ACC GAA TAT GCT CCC AAGTTC CAG GGC AAA GCT ACT ATG ACC GCT GAC ACC TCT AGT AAC ACT GCC TAC CTG CAGTTG AGC TCT CTT ACG TCT GAG GAT ACC GCT GTG TAC TAC TGT AAT AAC GGA AAT TATGTA CGA CAC TAT TAC TTC GAC TAC TGG GGG CAG GGC ACT ACT GTG ACT GTA TCTAGCtCTAGA

[0217] CD5 - 17H2L - 3054 CAR (SEQ ID NO:13)

[0218] ggatccGAGGTCCAACTCGTAGAATCAGGTCCCGGATTGGTGCAACCATCCCAGAGCCTCTCTATTACATGCACGGTCTCTGGATTTAGTCTGACCAATTACGATGTGCATTGGGTGCGCCAGTCTCCCGGCAAGGGGTTGGAATGGCTTGGCGTTATATGGAACTACGGAAATACAGACTATAACGCCGCGTTTATCTCTCGGCTGAGTATACGGAAAGACAGTAGTAAATCCCAGGTCTTTTTTACGATGTCATCCCTGCAAACGCCAGATACCGCAATATATTACTGCGCCAGGAACCACGGTGATGGTTATTATAATTGGTACTTCGATGTGTGGGGTACTGGCACTACAGTCACAGTATCTTCAggtggcggagggagcggcggtggaggaagcggaggcggaggttccAACATTGTACTGACGCAAAGCCCCTCATCTTTGTCTGAGTCACTCGGCGGCAAAGTAACCATCACATGCAAGGCCAGTCAAGACATCAATAAATATATTGCTTGGTATCAGTATAAACCCGGCAAGGGGCCGCGACTGCTGATTCACTACACGAGTACCTTGCAACCGGGCATTCCGAGCCGATTTAGTGGCAGTGGCTCAGGTCGCGATTACTCATTCTCAATAAGTAATCTCGAACCGGAAGACATAGCTACTTATTATTGCTTGCAGTACGATAATTTGTGGACCTTCGGGGGTGGTACAAAGTTGGAAATAAAGtctaga

[0219] CD8 transmembrane domain nucleic acid sequence (SEQ ID NO:14):

[0220] atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatc

[0221] accctttactgc

[0222] CD8 transmembrane domain amino acid sequence (SEQ ID NO:15):

[0223] IYIWAPLAGTCGVLLLSLVITLYC

[0224] CD8 hinge domain nucleic acid sequence (SEQ ID NO:16):

[0225] accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctg

[0226] tccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctg

[0227] gacttcgcctgtgat

[0228] CD8 hinge domain amino acid sequence (SEQ ID NO:17):

[0229] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0230] 4 - 1BB nucleic acid sequence (SEQ ID NO:18)

[0231] aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaa

[0232] actactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgt

[0233] gaactg

[0234] CD3-ζ nucleic acid sequence (SEQ ID NO:19)

[0235] agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctc

[0236] tataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggc

[0237] cgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaat

[0238] gaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgc

[0239] cggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacc

[0240] tacgacgcccttcacatgcaggccctgccccctcgc

[0241] 4 - 1BB amino acid sequence (SEQ ID NO:20) :

[0242] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0243] CD3 - ζ Amino acid sequence (SEQ ID NO:21):

[0244] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0245] CD2 - MEDI507H2L - 3028 CAR amino acid sequence (SEQ ID NO:25)

[0246] MALPVTALLLPLALLLHAARPGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0247] CD2 - MEDI507L2H - 3043 CAR amino acid sequence (SEQ ID NO:26)

[0248] MALPVTALLLPLALLLHAARPGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0249] CD2 - MEDI507H2L - 3028 scFv amino acid sequence (SEQ ID NO:27)

[0250] GSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKSG

[0251] CD2 - MEDI507L2H - 3043 scFv amino acid sequence (SEQ ID NO:28)

[0252] GSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSSSG

[0253] CD2 - MEDI507 VH amino acid sequence (SEQ ID NO:29)

[0254] QVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQGTLVTVSS

[0255] CD2 - MEDI507 VL amino acid sequence (SEQ ID NO:30)

[0256] DVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIK

[0257] CD2 - MEDI507 HCDR1 (SEQ ID NO:31)

[0258] EYYMY

[0259] CD2-MEDI507 HCDR2 (SEQ ID NO:32)

[0260] RIDPEDGSIDYVEKFKK

[0261] CD2-MEDI507 HCDR3 (SEQ ID NO:33)

[0262] GKFNYRFAY

[0263] CD2-MEDI507 LCDR1 (SEQ ID NO:34)

[0264] RSSQSLLHSSGNTYLN

[0265] CD2-MEDI507 LCDR2 (SEQ ID NO:35)

[0266] LVSKLES

[0267] CD2-MEDI507 LCDR3 (SEQ ID NO:36)

[0268] MQFTHYPYT

[0269] CD2-OKT11H2L-3029 CAR amino acid sequence (SEQ ID NO:37)

[0270] MALPVTALLLPLALLLHAARPGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0271] CD2-OKT11L2H-3030 CAR amino acid sequence (SEQ ID NO:38)

[0272] MALPVTALLLPLALLLHAARPGSDIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0273] CD2-OKT11H2L-3029 scFv amino acid sequence (SEQ ID NO:39)

[0274] GSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIKSG

[0275] CD2-OKT11L2H-3030 scFv amino acid sequence (SEQ ID NO:40)

[0276] GSDIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSSSG

[0277] CD2-OKT11 VH amino acid sequence (SEQ ID NO:41)

[0278] QVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTTLTVSS

[0279] CD2-OKT11 VL amino acid sequence (SEQ ID NO:42)

[0280] DIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIK

[0281] CD2-OKT11 HCDR1 (SEQ ID NO:43)

[0282] SYWMH

[0283] CD2-OKT11 HCDR2 (SEQ ID NO:44)

[0284] RIDPYDSETHYNEKFKD

[0285] CD2-OKT11 HCDR3 (SEQ ID NO:45)

[0286] RDAKYDGYALDY

[0287] CD2-OKT11 LCDR1 (SEQ ID NO:46)

[0288] RSSKTLLHSNGNTYLY

[0289] CD2-OKT11 LCDR2 (SEQ ID NO:47)

[0290] RMSNLAS

[0291] CD2-OKT11 LCDR3 (SEQ ID NO:48)

[0292] MQHLEYPYT

[0293] CD2-T11-2-H2L-3031 CAR amino acid sequence (SEQ ID NO:49)

[0294] MALPVTALLLPLALLLHAARPGSQVQLQQPGAELVRPGASVKLSCKASGYTFTTFWMNWVKQRPGQGLEWIGMIDPSDSEAHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGRGYDDGDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMQFTHYPYTFGGGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0295] CD2-T11-2-H2L-3031 scFv amino acid sequence (SEQ ID NO:50)

[0296] GSQVQLQQPGAELVRPGASVKLSCKASGYTFTTFWMNWVKQRPGQGLEWIGMIDPSDSEAHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGRGYDDGDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMQFTHYPYTFGGGTKLEIKSG

[0297] CD2-T11-2-H2L-3031 VH amino acid sequence (SEQ ID NO:51)

[0298] QVQLQQPGAELVRPGASVKLSCKASGYTFTTFWMNWVKQRPGQGLEWIGMIDPSDSEAHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGRGYDDGDAMDYWGQGTSVTVSS

[0299] CD2-T11-2-H2L-3031 VL amino acid sequence (SEQ ID NO:52)

[0300] DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMQFTHYPYTFGGGTKLEIK

[0301] CD2-T11-2 HCDR1 (SEQ ID NO:53)

[0302] TFWMN

[0303] CD2-T11-2 HCDR2 (SEQ ID NO:54)

[0304] MIDPSDSEAHYNQMFKD

[0305] CD2-T11-2 HCDR3 (SEQ ID NO:55)

[0306] GRGYDDGDAMDY

[0307] CD2-T11-2 LCDR1 (SEQ ID NO:56)

[0308] RASKSVSTSGYSYMH

[0309] CD2-T11-2 LCDR2 (SEQ ID NO:57)

[0310] LVSNLES

[0311] CD2-T11-2 LCDR3 (SEQ ID NO:58)

[0312] MQFTHYPYT

[0313] CD2-TS2-18.1.1-H2L-3032 CAR amino acid sequence (SEQ ID NO:59)

[0314] MALPVTALLLPLALLLHAARPGSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0315] CD2-TS2-18.1.1-L2H-3033 CAR amino acid sequence (SEQ ID NO:60)

[0316] MALPVTALLLPLALLLHAARPGSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0317] CD2-TS2-18.1.1-H2L-3032 scFv Amino Acid Sequence (SEQ ID NO:61)

[0318] GSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKSG

[0319] CD2-TS2-18.1.1-L2H-3033 scFv Amino Acid Sequence (SEQ ID NO:62)

[0320] GSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSSSG

[0321] CD2-TS2-18.1.1 VH Amino Acid Sequence (SEQ ID NO:63)

[0322] EVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTTLTVSS

[0323] CD2-TS2-18.1.1 VL Amino Acid Sequence (SEQ ID NO:64)

[0324] DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIK

[0325] CD2-TS2-18.1.1 HCDR1 (SEQ ID NO:65)

[0326] SYDMS

[0327] CD2-TS2-18.1.1 HCDR2 (SEQ ID NO:66)

[0328] YISGGGFTYYPDTVKG

[0329] CD2-TS2-18.1.1 HCDR3 (SEQ ID NO:67)

[0330] QGANWELVY

[0331] CD2-TS2-18.1.1 LCDR1 (SEQ ID NO:68)

[0332] RASQSISDFLH

[0333] CD2-TS2-18.1.1 LCDR2 (SEQ ID NO:69)

[0334] YASQSIS

[0335] CD2-TS2-18.1.1 LCDR3 (SEQ ID NO:70)

[0336] QNGHNFPPT

[0337] CD5-17L2H-3045 CAR Amino Acid Sequence (SEQ ID NO:71)

[0338] MALPVTALLLPLALLLHAARPGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSSRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0339] CD5-17H2L-3054 CAR Amino Acid Sequence (SEQ ID NO:72)

[0340] MALPVTALLLPLALLLHAARPGSEVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIKSRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0341] CD5-17L2H-3045 scFv Amino Acid Sequence (SEQ ID NO:73)

[0342] NIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSS

[0343] CD5-17H2L-3054 scFv Amino Acid Sequence (SEQ ID NO:74)

[0344] EVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIK

[0345] CD5-17 VH Amino Acid Sequence (SEQ ID NO:75)

[0346] EVQLVESGPGLVQPSQSLSITCTVSGFSLTNYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQTPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSS

[0347] CD5-17 VL Amino Acid Sequence (SEQ ID NO:76)

[0348] NIVLTQSPSSLSESLGGKVTITCKASQDINKYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIK

[0349] CD5-9H2L-3048 CAR Amino Acid Sequence (SEQ ID NO:77)

[0350] MALPVTALLLPLALLLHAARPGSQVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSHIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0351] CD5-9L2H-3049 CAR Amino Acid Sequence (SEQ ID NO:78)

[0352] MALPVTALLLPLALLLHAARPGSHIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0353] CD5-9H2L-3048 scFv Amino Acid Sequence (SEQ ID NO:79)

[0354] GSQVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSHIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKSG

[0355] CD5-9L2H-3049 scFv Amino Acid Sequence (SEQ ID NO:80)

[0356] HIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSS

[0357] CD5-9 VH Amino Acid Sequence (SEQ ID NO:81)

[0358] QVQLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSS

[0359] CD5-9 VL Amino Acid Sequence (SEQ ID NO:82)

[0360] HIVLTQSPSSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIK

[0361] CD5-9 HCDR1 (SEQ ID NO:83)

[0362] AYSMN

[0363] CD5-9 HCDR2 (SEQ ID NO:84)

[0364] SIDPYYGDTKYAQKFKG

[0365] CD5-9 HCDR3 (SEQ ID NO:85)

[0366] RMITTGDWYFDV

[0367] CD5-9 LCDR1 (SEQ ID NO:86)

[0368] RASQDISTYLN

[0369] CD5-9 LCDR2 (SEQ ID NO:87)

[0370] YTSRLHA

[0371] CD5-9 LCDR3 (SEQ ID NO:88)

[0372] QQGNSLPFT

[0373] CD5-34H2L-3052 CAR Amino Acid Sequence (SEQ ID NO:89)

[0374] MALPVTALLLPLALLLHAARPGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKSRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0375] CD5-34L2H-3053 CAR Amino Acid Sequence (SEQ ID NO:90)

[0376] MALPVTALLLPLALLLHAARPGSDWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSSSRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0377] CD5-34H2L-3052 scFv Amino Acid Sequence (SEQ ID NO:91)

[0378] EVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIK

[0379] CD5-34L2H-3053 scFv Amino Acid Sequence (SEQ ID NO:92)

[0380] DWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSS

[0381] CD5-34 VH Amino Acid Sequence (SEQ ID NO:93)

[0382] EVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWGQGTTVTVSS

[0383] CD5-34 VL Amino Acid Sequence (SEQ ID NO:94)

[0384] DWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKSR

[0385] CD5-34 HCDR1 (SEQ ID NO:95)

[0386] DYYIH

[0387] CD5-34 HCDR2 (SEQ ID NO:96)

[0388] WIDPENGRTEYAPKFQG

[0389] CD5-34 HCDR3 (SEQ ID NO:97)

[0390] GNYVRHYYFDY

[0391] CD5-34 LCDR1 (SEQ ID NO:98)

[0392] RAISSVSYMH

[0393] CD5-34 LCDR2 (SEQ ID NO:99)

[0394] ATSNLAS

[0395] CD5-34 LCDR3 (SEQ ID NO:100)

[0396] QQWSSNPRT

[0397] CRISPR / Cas

[0398] Certain embodiments of the present invention include cells that have been modified by a CRISPR / Cas system. The CRISPR / Cas system includes, but is not limited to, the CRISPR / Cas9 system and the CRISPR / Cpf1 system. In certain embodiments, the present invention includes cells that have been modified using the CRISPR / Cas9 system. In certain embodiments, the modification includes knocking out or mutating an endogenous gene, e.g., CD2, CD5, or CD7.

[0399] The CRISPR / Cas9 system is a simple and efficient system for inducing targeted gene 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 co-activation of target genes.

[0400] An example of a CRISPR / Cas system for inhibiting gene expression, CRISPRi, is described in U.S. Publication No. US2014 / 0068797, which is incorporated herein by reference in its entirety. CRISPRi induces permanent gene disruption, which utilizes an RNA-guided Cas9 endonuclease to introduce DNA double-strand breaks, which trigger error-prone repair pathways, resulting in frameshift mutations. 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 the expression of target genes.

[0401] 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 capable of enabling the Cas endonuclease to introduce a double-strand break at the target gene. In certain embodiments, the CRISPR system comprises an expression vector, such as but not limited to the pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces the expression of the Cas9 endonuclease. Other endonucleases can also be used, including but not limited to Cpf1, T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleases known in the art, and any combination thereof.

[0402] In certain embodiments, inducing the Cas expression vector comprises exposing the cell to a reagent that activates an inducible promoter in the Cas expression vector. In such an embodiment, the Cas expression vector comprises an inducible promoter, such as a promoter that can be induced by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, e.g., doxycycline). However, it should be understood that other inducible promoters can be used. The inducer can be a selective condition that results in induction of the inducible promoter (e.g., exposure to a reagent, such as an antibiotic). This results in the expression of the Cas expression vector.

[0403] The guide nucleic acid sequence is specific for a gene and targets that gene for Cas endonuclease-induced double-strand break. 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.

[0404] 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 having 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.

[0405] In the context of forming a CRISPR complex, a "target sequence" is a sequence to which the guide sequence is designed to have some complementarity, where hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and facilitate the formation of the CRISPR complex. The target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence can be within an organelle of a eukaryotic cell (e.g., mitochondrion or nucleus). 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 the target sequence (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs). As with the target sequence, perfect complementarity is believed not to be required, as long as this is sufficient to function. In some embodiments, when optimally aligned, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence.

[0406] In other embodiments, one or more vectors that drive the expression of one or more elements of the CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system directs the 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. Optionally, two or more of the elements expressed by 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 CRISPR system elements combined in a single vector can be arranged in any suitable orientation, such as one element located 5' relative to a second element ("upstream") or 3' relative to a second element ("downstream"). The coding sequence of one element can be on the same or opposite strand of the coding sequence of the second element and be oriented in the same or opposite direction. In some embodiments, a single promoter drives the 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 within 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).

[0407] In certain embodiments, the CRISPR enzyme is part of a fusion protein that includes 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 the CRISPR enzyme). The CRISPR enzyme fusion protein can include any additional protein sequences, as well as optionally a linker sequence between any two domains. Examples of protein domains that can be fused to the 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 containing the CRISPR enzyme are described in US20110059502, which is incorporated herein by reference. In certain embodiments, the tagged CRISPR enzyme is used to identify the location of the target sequence.

[0408] Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into 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 a host organism. 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 delivery mode for CRISPR / Cas9 includes a combination of RNA and purified Cas9 protein, which exists in the form of a Cas9-guide RNA ribonucleoprotein (RNP) complex. (Lin et al., 2014, eLife 3:e04766). Viral vector delivery systems include DNA and RNA viruses that have episomal or integrated genomes after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu et al., 1994, Gene Therapy 1:13-26).

[0409] 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, Cpf1, CasY, CasX, or saCas9.

[0410] Generally, a CRISPR / Cas protein comprises at least one RNA recognition and / or RNA binding domain. The RNA recognition and / or RNA binding domain interacts with a guide RNA. The CRISPR / Cas protein may further comprise a nuclease domain (i.e., a DNA nuclease or an RNA nuclease domain), a DNA binding domain, a helicase domain, an RNA nuclease 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, alter enzyme activity, and / or 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.). Optionally, domains of the Cas9 protein that do not participate in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild-type Cas9 protein. Generally, the Cas9 protein comprises at least two nuclease (i.e., DNA nuclease) domains. For example, the Cas9 protein can comprise a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains act together to cleave single strands, thereby creating a double-strand 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 (a RuvC-like or an 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 no longer functions (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, as well as other methods known in the art.

[0411] In a non-limiting embodiment, a vector drives the expression of the CRISPR system. The art is full of suitable vectors for use in the present invention. The vectors used are suitable for replication and, optionally, integration into eukaryotic cells. Typical vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequences. The vectors of the present invention can also be used in nucleic acid standard gene delivery protocols. Methods of gene delivery are known in the art (U.S. Patent Nos. 5,399,346, 5,580,859 & 5,589,466, which are incorporated herein by reference in their entirety).

[0412] Further, the vector can be provided to the cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4 th 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, adeno-associated viruses, herpesviruses, Sindbis viruses, gamma-retroviruses, and lentiviruses. Generally, suitable vectors contain 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., WO01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0413] Introduction of Nucleic Acid

[0414] Methods for introducing nucleic acids into cells include physical, biological, and chemical methods. Physical methods for introducing polynucleotides (such as 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 lipid-mediated lipofection, using polymer encapsulation, using peptide-mediated transfection, or using a biological projectile particle delivery system such as a "gene gun" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

[0415] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially 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 lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0416] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0417] Suitable lipids are available from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("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 approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term that encompasses a variety of single and multi-layer lipid carriers formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized by having a vesicular structure containing a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. Before forming a closed structure, the lipid components undergo self-rearrangement and trap water and dissolved solutes between the lipid bilayers (Ghosh et al., 191 Glycobiology 5; 505-10). However, also included are compositions that have a different structure in solution compared to the normal vesicular structure. For example, the lipids can assume a micellar structure or exist only as heterogeneous aggregates of lipid molecules. Also contemplated are lipid transfection amine-nucleic acid complexes.

[0418] Regardless of the method used to introduce exogenous nucleic acid into a host cell, or otherwise expose the cell to an inhibitor of the invention, in order to confirm the presence of nucleic acid in the host cell, a variety of assays can be performed. Such assays include, for example, "molecular biology" assays well known to those of skill in the art, such as Southern and Northern blots, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a specific peptide, for example, by immunological means (ELISA and Western blot) or by assays that recognize reagents falling within the scope of the invention described herein.

[0419] In addition, nucleic acid can be introduced by any means, such as transducing expanded T cells, transfecting expanded T cells, and electroporating expanded T cells. One nucleic acid can be introduced by one method, while another nucleic acid can be introduced into T cells by a different method.

[0420] RNA

[0421] In one embodiment, the nucleic acid introduced into the T cells is RNA. In another embodiment, the RNA is mRNA comprising in vitro transcribed RNA or synthetic RNA. The RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). The DNA of interest from any source can be directly converted into a template by PCR for use with appropriate primers and RNA polymerase for in vitro mRNA synthesis. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source. The desired template for in vitro transcription is a chimeric membrane protein. By way of example, the template encodes an antibody, an antibody fragment, or a portion of an antibody. By another example, the template comprises an extracellular domain that includes a single-chain variable domain of an antibody, such as anti-CD3, and an intracellular domain of a co-stimulatory molecule. In one embodiment, the template for the RNA chimeric membrane protein encodes a chimeric membrane protein that comprises an extracellular domain and an intracellular domain, the extracellular domain comprising an antigen-binding domain of an antibody derived from a co-stimulatory molecule, and the intracellular domain being derived from a portion of the intracellular domains of CD28 and 4-1BB.

[0422] PCR can be used to generate a template for in vitro transcription of mRNA, which is then introduced into cells. Methods for performing PCR are well known in the art. The primers for PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as the template for PCR. As used herein, "substantially complementary" refers to a sequence of nucleotides where most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. A substantially complementary sequence is capable of annealing or hybridizing to the desired DNA target under the annealing conditions used for PCR. The primers can be designed to be substantially complementary to any part of the DNA template. For example, the primers can be designed to amplify a portion (open reading frame) of a gene that is normally transcribed in cells, including the 5' and 3' UTRs. The primers can also be designed to amplify a portion of a gene encoding a specific domain of interest. In one embodiment, the primers are designed to amplify the coding region of human cDNA, including all or part of the 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 nucleotide region that is substantially complementary to the nucleotides on the DNA template upstream of the DNA sequence to be amplified. As used herein, "upstream" refers to the position 5' relative to the DNA sequence to be amplified on the coding strand. A "reverse primer" is a primer that contains a nucleotide region that is substantially complementary to the downstream double-stranded DNA template of the DNA sequence to be amplified. As used herein, "downstream" refers to the position 3' relative to the DNA sequence to be amplified on the coding strand.

[0423] Chemical structures having the ability to enhance RNA stability and / or translation efficiency can also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR has a length of 0 to 3000 nucleotides. The lengths of the 5' and 3' UTR sequences added to the coding region can be altered by different methods (including but not limited to designing PCR primers that anneal to different regions of the UTR). Using this method, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0424] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Optionally, 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. Using UTR sequences that are not endogenous to the gene of interest can be used to modify RNA stability and / or translation efficiency. For example, AU-rich elements in the 3' UTR sequence are known to reduce mRNA stability. Thus, the 3' UTR can be selected or designed based on the properties of UTRs well known in the art to increase the stability of the transcribed RNA.

[0425] In one embodiment, the 5' UTR can contain the Kozak sequence of an endogenous gene. Optionally, when a 5' UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. The Kozak sequence can increase the translation efficiency of some RNA transcripts, but does not appear to be required for all RNAs to be translated efficiently. The requirement for the Kozak sequence 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 for the 3' or 5' UTR to prevent exonucleolytic degradation of the mRNA.

[0426] In order to be able to synthesize RNA from a DNA template without gene cloning, a transcription promoter should be ligated to the DNA template upstream of the sequence to be transcribed. When a sequence that serves as a promoter for 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 the T7 polymerase promoter as described elsewhere herein. Other useful promoters include but are not limited to the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0427] In one embodiment, the mRNA has a cap on both the 5' end and the 3' poly(A) tail, which determines ribosome binding, the initiation of translation, and the stability of the mRNA in the cell. On a circular DNA template (e.g., plasmid DNA), RNA polymerase produces a long concatameric product, which is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end produces a normal-sized mRNA, which is not effective in eukaryotic transfection even after being polyadenylated post-transcriptionally.

[0428] On a linear DNA template, 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).

[0429] The conventional method of integrating a polyA / T sequence into a DNA template is molecular cloning. However, the polyA / T sequence integrated into plasmid DNA can lead to 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 also often unreliable. This is why a method of constructing a DNA template with a polyA / T 3' sequence without the need for cloning is highly desirable.

[0430] The polyA / T fragment of the transcribed DNA template can be generated during PCR by using a reverse primer containing a polyT tail (such as a 100T tail (the size can be 50 - 5000T)), or by any other method after PCR (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. Generally, 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.

[0431] After in vitro transcription, the poly(A) tail of the RNA can be further extended using a poly(A) polymerase such as Escherichia 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 a approximately two-fold increase in the translation efficiency of the RNA. Additionally, attaching different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified / artificial nucleotides, aptamers, and other compounds. For example, an ATP analog can be incorporated into the poly(A) tail using a poly(A) tail polymerase. The ATP analog can further enhance the stability of the RNA.

[0432] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein includes 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)).

[0433] 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 ribosome binding to the mRNA and promotes the initiation of translation. Any suitable solute 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.

[0434] In some embodiments, the RNA (such as in vitro transcribed RNA) is electroporated into cells.

[0435] The disclosed methods can be applied to the regulation of T cell activity in basic research and therapies 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.

[0436] The method also provides the ability to control the expression level over a wide range by altering, for example, the promoter or the amount of input RNA, such that the expression level can be regulated individually. Additionally, PCR-based mRNA production technology has greatly facilitated the design of mRNAs with different structures and their domain combinations.

[0437] One advantage of the RNA transfection method of the present invention is that RNA transfection is substantially transient and vector-free. The RNA transgene can be delivered and expressed in lymphocytes as a minimal expressing cassette without any additional viral sequences after a brief in vitro cell activation. Under these conditions, the likelihood 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, cell cloning is not necessary.

[0438] Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies that have been successively tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. Various modifications are desired to stabilize IVT-RNA to achieve extended expression of the transferred IVT-RNA.

[0439] Some IVT vectors are known in the literature and are used as templates for in vitro transcription in a standardized manner and have been genetically modified in a way that produces stable RNA transcripts. The current protocols used in the art are based on plasmid vectors having the following structure: a 5' RNA polymerase promoter capable of performing RNA transcription; followed by a gene of interest flanked by untranslated regions (UTRs) 3' and / or 5'; and a 3' polyadenylation cassette containing 50 - 70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette by a type II restriction enzyme (the recognition sequence corresponding 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 affects the amount of protein produced intracellularly from this construct.

[0440] Compared to more traditional plasmid or viral methods, RNA has several advantages. Gene expression from an RNA source does not require transcription and protein products are rapidly produced after transfection. Further, since RNA can only enter the cytoplasm, not the nucleus, typical transfection methods result in extremely high transfection rates. In addition, plasmid-based methods require that the promoter driving the expression of the gene of interest be active in the cells under study.

[0441] In another aspect, the RNA construct is delivered into cells by electroporation. See, for example, the 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. In the relevant research literature as well as in numerous patents and applications in the field, various parameters are generally known, which include the electric field strength required for electroporation of 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 the therapeutic application of electroporation are commercially available, for example, the MedPulser TM DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and are 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 for in vitro transfection of cells, as described, for example, in US20070128708A1. Electroporation can also be used to deliver nucleic acids into cells in vitro. Thus, electroporation-mediated administration of nucleic acids, including expression constructs, into cells using any of the various available devices and electroporation systems known to those of skill in the art provides an exciting new means for delivering the RNA of interest to target cells.

[0442] Source of T Cells

[0443] In certain embodiments, a T cell source 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, which include T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis can be washed to remove the plasma portion and the cells can be placed in an appropriate buffer or medium, such as phosphate buffered saline (PBS), or a wash solution 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, such as Ca-free, Mg-free PBS. Optionally, unwanted components of the apheresis sample can be removed and the cells can be directly resuspended in a culture medium.

[0444] In another embodiment, for example, T cells are isolated from peripheral blood by PERCOLL TM gradient centrifugation, by lysing red blood cells and depleting monocytes. Optionally, T cells can be isolated from umbilical cord. In any case, specific subsets of T cells can be further isolated by positive or negative selection techniques.

[0445] 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. These cell depletions can be accomplished using isolated antibodies, biological samples containing antibodies, such as ascites, antibodies conjugated to a physical support, and cell-bound antibodies.

[0446] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed against surface markers unique to the negatively selected cells. Preferred methods are via negative magnetic immunoadherence or flow cytometry, which uses a mixture of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture generally includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0447] To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may 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 between the cells and the 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, a concentration greater than 100 million cells / ml is used. In further embodiments, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml is used. In further embodiments, a concentration of 125 million or 150 million cells / ml can be used. Using a high concentration can result in increased cell yield, cell activation and cell expansion.

[0448] T cells can also be frozen after the washing step, which does not require a monocyte removal step. Although not wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and to some extent monocytes from the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Although a variety of freezing solutions and parameters are known in the art and will be useful in the context, in a non-limiting example, one method involves using 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 immediately at -20°C or in liquid nitrogen.

[0449] In one embodiment, the T cell population is contained within cells such as peripheral blood mononuclear cells, cord blood cells, purified T cell populations and T cell lines. In another embodiment, peripheral blood mononuclear cells contain the T cell population. In yet another embodiment, purified T cells contain the T cell population.

[0450] Expansion of T Cells

[0451] 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 the range of about 20-fold to about 50-fold.

[0452] 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 passage, before transferring the cells to another culture device. The culture device can be any culture device commonly used for in vitro cell culture. Preferably, the confluence level is 70% or higher before transferring the cells to another culture device. More preferably, the confluence level is 90% or higher. The period of time can be any time suitable for in vitro cell culture. The T cell culture medium can be changed at any time during the culture of the T cells. Preferably, the T cell culture medium is changed approximately every 2 - 3 days. The T cells are then harvested from the culture device and can subsequently be used immediately or cryopreserved for later use. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed before introducing the nucleic acid into the T cells.

[0453] In another embodiment, the method includes isolating T cells and expanding the T cells. In another embodiment, the invention further includes cryopreserving the T cells before expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding a chimeric membrane protein.

[0454] Another procedure for ex vivo expansion of cells is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). The expansion (as described in U.S. Patent No. 5,199,942) can be an alternative or supplement to other expansion methods described herein. Briefly, the ex vivo culturing and expansion of T cells includes adding 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 flt3-L, IL-1, IL-3, and c-kit ligand.

[0455] The culturing steps (after contact with the reagents or electroporation as described herein) can be very short, e.g., 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 (contact with the reagents as described herein) can be longer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days, as further described herein.

[0456] A variety of 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 before the first passage. When cells are placed in a growth medium under conditions that promote cell growth and / or division, the cells expand in culture, resulting in a larger cell population. When cells expand in culture, the cell proliferation rate is generally measured by the amount of time required for the cell number to double (also referred to as the doubling time).

[0457] Each round of passage is called a passage. When cells are passaged, they are said to have been passaged. A particular cell population or cell line is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged 10 times can be called a P10 culture. A primary culture (i.e., the first culture after cells are isolated from tissue) is called P0. After the first passage, the cells are described as a secondary culture (P1 or passage 1). After the second passage, the cells become a third-generation culture (P2 or passage 2), and so on. Those skilled in the art will understand that there can be multiple population doublings during a passage; thus, the population doubling number of a culture is greater than the passage number. 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 the time between passages.

[0458] In one embodiment, the cells can be cultured for several hours (about 3 hours) to about 14 days or any whole hour value therebetween. Conditions suitable for T cell culture include a suitable culture medium (e.g., Minimal Essential Media or RPMI Medium 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-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-α 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. The culture medium can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free 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 and not in cell cultures to be infused into a subject. The target cells are maintained under conditions required for growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0459] The culture medium for culturing T cells can include reagents that can co-stimulate T cells. For example, a reagent that can stimulate CD3 is an antibody against CD3, and a reagent that can stimulate CD28 is an antibody against CD28. This is because, as demonstrated by the data disclosed herein, the cells isolated by the methods disclosed herein can be expanded 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 greater. In one embodiment, the T cells are expanded in the range of about 20-fold to about 50-fold or more by culturing the electroporated population.

[0460] In one embodiment, the method of expanding T cells can further include isolating the expanded T cells for further use. In another embodiment, the expansion method can further include subsequently electroporating the expanded T cells, followed by culturing. The subsequent electroporation can include introducing a nucleic acid encoding a reagent 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 reagent further stimulates the T cells. The reagent can stimulate the T cells, for example, by stimulating further expansion, effector function, or another T cell function.

[0461] Drug Composition

[0462] The pharmaceutical compositions of the present invention 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 dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0463] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The quantity and frequency of administration will be determined by factors such 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.

[0464] For a subject receiving therapy, the cells of the present invention to be administered can be autologous, allogeneic, or xenogeneic.

[0465] The cells of the present invention can be administered at dosages, via routes, and at times to be determined in appropriate preclinical and clinical experiments and trials. The cell compositions can be administered multiple times at dosages within these ranges. The administration of the cells of the present invention can be combined with other methods determined by those skilled in the art for treating the desired disease or condition.

[0466] It can generally be stated that pharmaceutical compositions comprising the T cells described herein can be administered at 10 4 to 10 9 cells / kg body weight, and in some cases 10 5 to 10 6administered at a dose of cells / kg body weight - including all integer values within those ranges - and the T cell compositions can also be administered multiple times at these doses. The cells can be administered by using infusion techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by one of ordinary skill in the medical arts by monitoring the patient's signs of disease and thereby adjusting the treatment.

[0467] The administration of the modified cells of the invention can be carried out in any convenient manner known to those skilled in the art. The cells of the invention can be administered to a subject by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient by arterial, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intravenous (i.v.) injection, or intraperitoneal injection. In other instances, the cells of the invention are directly injected into the site of inflammation of the subject, the local disease site of the subject, lymph nodes, organs, tumors, and the like.

[0468] It should be understood that the methods and compositions useful in the present invention are not limited to the specific formulations set forth in the examples. The following examples are presented to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the cells, expansion and culture methods, and treatment methods of the present invention and are not intended to limit the scope of what the inventors regard as their invention.

[0469] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained 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 present invention and, accordingly, can be considered in the manufacture and practice of the present invention. Particularly useful techniques for specific embodiments will be discussed in the following sections.

[0470] Experimental Examples

[0471] The present invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only and the present invention is not limited to these examples, but encompasses all variations that are apparent from the teachings provided herein.

[0472] The materials and methods used in these experiments will now be described.

[0473]

[0474] 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. The 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 by electroporation. The expression of CD2 / CD5 / CD7 on Jurkat cells was detected by flow cytometry, and the most effective CD2 / CD5 / CD7 sgRNAs were identified. Then, CRISPR editing was performed in primary human T cells using the most effective CD2 / CD5 / CD7 sgRNAs: the selected sgRNAs and Cas9 RNA were electroporated into primary human T cells. CD2 / CD5 / CD7 expression was detected on primary human T cells by flow cytometry to verify the knockout / editing efficiency.

[0475] Specifically, fresh CD4 / CD8 T cells were obtained on day 0 and incubated with dynabeads. On day 4, the cells were bead-depleted 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 evaluated. When the volume < 300 fl, the cells were fed to 0.8e6 / ml and frozen ( Figure 22 ).

[0476] CAR constructs: All constructs were generated using the lentiviral pTRPE 4-1BB CD3ζ backbone. The OKT11 CAR and TS2 / 18.1.1 CAR were constructed using scFvs derived from hybridomas purchased from ATCC ( CRL-8027 TM and HB-195 TM ), respectively). The T11-2 CAR was constructed using an scFv derived from an antibody produced by a hybridoma received from Ellis Reinherz. All CD5 CARs were constructed using scFvs from antibody sequences disclosed in WO 2010 / 022737A1, the content of which is incorporated herein by reference in its entirety.

[0477] The results of the experiments are now described.

[0478] Example 1: A New Method for Targeting T-Cell Lymphoma and Leukemia without Causing T-Cell Toxicity

[0479] The overall prognosis of T-cell lymphomas and leukemias is very poor, and there are few available treatment options for these patients. Chimeric antigen receptor T-cell (CAR T) immunotherapy has achieved unprecedented results in CD19+ B-cell non-Hodgkin lymphoma (B-NHL). In this article, another successful "CAR19-like" product was designed to target T-NHL. Since CD19 is not expressed in T-NHL, other targets such as CD2, CD5, CD7, etc. were evaluated for CAR T therapy. However, all of these targets are also expressed by normal T cells, resulting in unacceptable clinical toxicity (T-cell hypoplasia - immunodeficiency)( Figure 1 ). In this article, a safe and effective CAR T strategy for treating T-cell lymphoma was developed by editing normal T cells that are resistant to CAR killing( Figure 2 ). When the CAR T targets (CD2, CD5, CD7) are temporarily removed from normal T cells, CAR T therapy against T-cell lymphomas and leukemias is feasible, thus avoiding CAR-mediated killing and immunodeficiency( Figure 2 ).

[0480] Example 2: Anti-CD5 CAR T Cells (CART5) and CD5 Knockout (KO) Normal T Cells

[0481] This article discloses a two-pronged immunotherapy that includes anti-CD5 CAR T cells (CAR T5) and CD5 knockout (KO) normal T cells( Figure 3 ). CAR T5 destroys T-cell lymphoma (such as T-NHL) or T-cell leukemia cells, but also kills normal T cells. Infusion of CD5 KO normal T cells provides CAR-resistant T-cell immunity until the CAR T5 cells are depleted, in some cases by using suicide genes (such as iCasp9, CD20 / rituximab, or others).

[0482] Since CD5 is highly expressed on T-NHL cells and not expressed in other tissues except T cells and a small subset of B cells, CD5 was selected as the T-NHL target. Six anti-CD5 CAR constructs with different affinities (#17, #34, #9 with high, medium, and low affinities respectively (Klitgaard JL, et al. (2013) British journal of haematology 163:182-93)) were generated using single-chain variable fragments (scFv) and expressed in T cells( Figures 4-5)。Interestingly, although CD5 is expressed in 100% of CART cells, anti-CD5 CART does not require CD5 knockout to generate CART5 cells (Mamonkin M, et al. (2015) Blood 126:983-92), although CD5 expression is lower compared to control T cells ( Figure 8 )。In the absence of CD5 CRISPR-Cas9 KO, the mean fluorescence intensity (MFI) of CD5 in CART5 is 10-fold lower compared to control T cells, while another pan T cell marker such as CD2 remains unchanged ( Figure 8 )。

[0483] The in vitro and in vivo activities of different CART5 constructs were compared. The construct C3054 from the high-affinity scFv #17 showed the best in vivo killing. Jurkat cells were transduced with different CAR5 constructs ( Figure 13 , targeting epitopes and affinities are shown on the left) and the GFP-NFAT reporter gene, and then co-cultured with CD5+ tumor cells (or control) for 24 hours. The leading CART5 (C3054) showed increased NFAT activation ( Figure 13 )。

[0484] The leading anti-CD5 CART was implemented with a suicide system and its function was tested in vitro and in vivo. Without being bound by a specific theory, the removal of CD5 (CRISPR-Cas9 KO) further increases the anti-tumor effect of CART5 by eliminating possible cis-surface interactions between CAR5 and CD5 on CART5.

[0485] Insertion of a suicide system into the leading CART5 product: The leading candidate CART5 (C3054) product was engineered to express a suicide system ( Figure 26 )。A P2A bicistronic vector encoding CAR5 and the inducible-caspase9 suicide system (DiStasi A, et al. (2011) 365:1673-83) (iCART5) was developed. Two orientations were cloned, CAR5-P2A-iCasp9 and iCasp9-P2A-CAR5 ( Figure 26 ), to define the most effective one (higher percentage of double-expressing cells and lower CAR5+iCasp9-%, to ensure safety). The iCART5 was effectively eliminated using the clinical-grade compound rimiducid (AP1903, Bellicum Pharmaceuticals).

[0486] In vitro testing of iCART5: Newly generated iCART5 were compared with WT CART5 to confirm their efficacy (luciferase-based in vitro killing) and phenotype / function after antigen stimulation (CD5+Jurkat T leukemia cells) (flow cytometry phenotype, 30-plex cytokine analysis by Luminex assay, CFSE proliferation and CD107a 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 examining killing at 15', 30' and 2, 6, 12 and 24 hours. iCART5 was also tested against primary T-NHL cells by a killing assay established using primary CFSE-labeled Sezary cells.

[0487] In vivo testing of iCART5: An in vivo xenograft model (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 (50ug / mouse 23) to deplete iCART5 vs. WT CART5 in vivo. 2x10e 6 CART5 cells / mice were injected with NOD SCIDγ-deficient mice (NSG) mice (8 per group). Tumor burden over time was assessed as bioluminescence (CBG), and T cell phenotypes were studied by flow cytometry and amplified by bioluminescence (CBR) studies at multiple time points (hours / days). Mice were raised for long-term (3-4 months) for survival and monitoring recurrence. A human T-NHL xenograft model was previously established by iv injection of primary Sezary cells. This model is used to test the anti-tumor and exhaustion efficacy of iCART5.

[0488] Evaluation of the role of CD5 KO in CART5: Preliminary data indicate that in vivo, CD5 CART5 is more effective than WT CART5. CD5 was knocked out on CART5 cells using CRISPR-Cas9. The CRISPR-Cas9 CART expansion protocol was optimized previously. Wild-type CART5 was compared with CD5 KO CART5 in vitro by testing CART5 viability, antigen-driven proliferation (using CFSE labeling), cytokine production (by 30-plex Luminex), degranulation (evaluated by CD107a by flow cytometry), cytotoxicity (luciferase-based), and phenotype (memory subsets, Th1 / Th2). Cell lines (e.g., Jurkat) and primary samples were used as targets. In vivo comparison of CD5 KO vs. WT CART5 (1x10e6 cells / mouse) was performed in NSG mice carrying Jurkat, and expansion and phenotype in peripheral blood were monitored on days 10 and 14.

[0489] Mechanisms by which CART5 function is enhanced by CD5 KO: Since CD5KO has been shown to increase CART5 activity, additional studies were conducted to understand the mechanism: i. Confocal imaging to analyze the localization of CAR5 and CD5 on CART5 cells; ii. Single-molecule imaging (ONI nanoimager) demonstrated cis-binding of CAR5 to CD5; iii. Expression of CAR5 in CD5+ Jurkat and showing that CART5 failed to kill CAR5+ Jurkat because the CD5 epitope (by CAR5) was masked; 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.

[0490] Generation of CART-resistant normal T cells to avoid T cell hypoplasia: CART5 cannot distinguish neoplastic T cells from normal T cells because both express similar levels of CD5. CART-resistant normal T cells were developed for co-infusion with anti-T-NHL CART to ensure immunity during CART anti-tumor activity. CRISPR-Cas9 gene editing was used to knock out CD5 in normal T cells, rendering them invisible to CART5. An efficient CRISPR-Cas9 gRNA (#4) was generated using an optimized CART expansion protocol, which could KO approximately 95% of CD5 in normal T cells. Data showed that CD5 KO T cells were resistant to CART5, while WT T cells (CD5+) were vigorously killed within 24 hours.

[0491] Generation of CD5 KO normal T cells: CD5 KO normal T cells were developed using a highly efficient gRNA (#4), which was electroporated using Lonza 4D Nucleofector together with Cas9 protein (ThermoFisher v2). On day 1, CRISPR-Cas9 KO was performed, and then the cells were cultured at 30 °C for 2 days to increase gene editing, and then activated and expanded with anti-CD3 / CD28 Dynabeads (beads::1T cell) until they reached a cell volume < 300 fl.

[0492] In vitro evaluation of the resistance of CD5 KO normal T cells to iCART5 killing:

[0493] The 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 showed that CD5 KO conferred resistance. Three additional T cell donors will be tested.

[0494] In vivo evaluation of the resistance of CD5 KO normal T cells to iCART5 in an autologous xenograft model: NSG mice (8 mice / group) were implanted with luciferase + CD5 KO normal T cells or WT, and autologous iCART5 was injected two days later. The effects of iCART5 on CD5 ko and WT normal T cells were evaluated by bioluminescence. Once the WT T cells were completely eliminated by iCART5 (luminescence), rimiducid was administered to deplete iCART5. Then WT T cells were reinjected to demonstrate that normal T cells could repopulate the host. Mice were bled weekly to evaluate CART expansion.

[0495] Evaluation of the role of CD5 KO on normal T cell function: The role of CD5 KO in normal T cells was investigated by carefully studying T cell effector functions. After TCR-specific stimulation (anti-CD3 / CD28 beads), cytokine production (30-plex Luminex), proliferation (CFSE), and activation of CD5KO T cells vs. WT were measured. It was also tested whether CD5KO T cells proliferated and produced similarly to WT when exposed to common infections.

[0496] Defining the optimal CD5KO normal T cell dose for clinical use: To ensure sufficient T cell immunity against the most common infections, the minimum number of cells to be infused in relapsed or refractory (r / r) T-NHL patients was defined using in silico and experimental methods (TCR sequencing and tetramer staining of TCR specific for infectious pathogens).

[0497] In a phase 1 pilot clinical trial of 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.

[0498] Clinical trial protocol design: The phase 1 clinical trial included r / r T-NHL patients treated using a 3+3 protocol design. From a single apheresis procedure, 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 / m2 x 5 days)] and product #1. If no dose-limiting toxicity (DLT) was observed, cohort 2 received lymphodepletion, product #1, and 1-5x10e 7 Total iCART5 (product #2) (10%, 30%, 60% of the total dose); 1-5x10e 7 Total CART5 was based on the suboptimal dose from the CART19 experiment in B-NHL8. If no DLT was observed in cohort 2, cohort 3 received lymphodepletion, product #1, and 1-5x10e 8 Total CART5 (full dose). If no toxicity was observed within the first 4 weeks, patients in cohort 1# were allowed to enter cohort 2#. Based on the tumor clearance rate (and reaching a maximum at month 6), the iCART5 cells were depleted using the dimerizer rimiducid (NCT02744287) to prevent possible long-term T-cell toxicity.

[0499] Preparation of the IND package and FDA submission: In collaboration with the Clinical Vaccine and Cell Production Facility (CVPF), clinical-grade manufacturing was optimized. The results of all the preclinical experiments described were formatted together with the clinical trial protocol to fit the IND application. Extensive support was provided within the CCI and ACC for the preparation of the IND.

[0500] Patient Enrollment and Treatment: After successful submission of the IND and approval by all regulatory agencies, a Phase 1 trial within the Lymphoma Program at the University of Pennsylvania was initiated (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-phase studies. Dr. Ruella is the Principal Investigator of the trial, and Dr. Carl June is the Scientific Protocol Advisor. Manufacturing of both products was conducted at CVPF.

[0501] Associated Research: Patient samples (peripheral blood) were analyzed at multiple time points (apheresis, days 1, 0, 7, 14, 28, 60, 90) to test CAR T expansion (qPCR and flow cytometry), CAR T phenotype (CyTOF), CAR T gene expression profile (GEP) (NanoString, single-cell RNAseq 10X Genomics), and cytokine levels in serum (Luminex, 30-plex array). When available, additional studies were performed on tumor biopsies before and after treatment (RNAseq and Hyperion analysis of the tumor microenvironment).

[0502] This trial will be an important milestone in the development of novel combination immunotherapies 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 also inevitably kill normal T cells due to similar CD5 expression. However, the strategy described herein includes co-infusion of normal T cells in which CD5 has been knocked out, thus ensuring T-cell immune protection during CAR T5 anti-tumor activity. A suicide system is then used to deplete CAR T5 cells to ensure long-term normal immune reconstitution. This is one of the first CAR T trials for T-NHL and the only one that includes a two-pronged approach to address toxicity. The prognosis for T-NHL is poor, and there are currently no available active immunotherapies. 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 associated research, this strategy could also be applied simultaneously to target multiple targets to avoid antigen-loss escape (e.g., CAR T5 + CAR T7) or combine CAR T with small molecules that enhance CAR T-mediated killing.

[0503] Example 3: Anti-CD2 CAR T Cells (CART2) and CD2 Knockout (KO) Normal T Cells

[0504] The present disclosure relates to a two-pronged immunotherapy method that includes anti-CD2 CAR T cells (CAR T2) and CD2 knockout (KO) normal T cells (Figure 3 ) CART2 destroys T cell lymphomas (such as T-NHL) or T cell leukemia cells, but 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 a suicide gene (such as iCasp9).

[0505] Guide RNAs were designed to knock out the CD2 gene (and CD5) using the CRISPR / Cas9 system. CD2 of 78% of the T cell population was effectively knocked out. Second-generation anti-CD2 and anti-CD5 CARs (CART2 and CART5 respectively) were generated ( Figure 4 ) The knocked-out cells (CD2KO and CD5KO) were incubated, stimulated and population doublings were measured together with their corresponding CART cells (CART2 and CART5 respectively) ( Figure 7 ) Blank control (Mock) electroporated cells without gRNA were used as a control for comparison. In the absence of CD2 KO, CART2 cells will not expand ( Figure 7 ) In the presence of KO CART2 and CART5, approximately 5 to 8 population doublings were achieved ( Figure 7 )

[0506] Jurkat cells were transduced with different CAR2 constructs and a GFP-NFAT reporter gene and then co-cultured with CD2+ tumor cells (or control) for 24 hours. Lead CART2 (C3043) showed increased NFAT activation ( Figure 14 )

[0507] The CART amplification protocol was optimized and CART2 and CART5 cells continued to expand up to 18 days when incubated with CD2KO or CD5 KO cells ( Figure 9 )

[0508] Example 4: CART2 and CART5 Tests

[0509] Figure 6 The CD5 (or CD2, or CD7) KO manufacturing process and CRISPR-Cas9 KO efficiency were exemplified.

[0510] Six different CAR2 and six CAR5 constructs were challenged in vitro by co-culturing them with luciferase+ Jurkat cells (T-cell leukemia cell line). At 24 hours, total killing was measured as the relative reduction in luminescence. For CART2, only #3029, #3030 and #3043 showed anti-tumor effects. ( Figure 10 ) For CART5, all constructs showed anti-tumor effects ( Figure 11)。The leading CART candidates (CART2 C3043 and CART5 C3054) were selected and tested. The effects of CD2 or CD5 knockout on CART function were tested). CART2- and CART5-resistant T cells were successfully generated (CD5 and CD2 were knocked out in normal T cells).

[0511] The activities of CART2 and CART5 against cutaneous T cell lymphoma were tested. A twenty-four-hour killing assay was performed. CART2 cells were active against primary Sezary cells (leukemic cutaneous T cell lymphoma) and the HH Sezary cell line ( Figure 15 ). CART5 was also active against HH cells ( Figure 15 ).

[0512] The in vivo efficacy of CART2 and CART5 was measured. NSG mice were implanted with luciferase+Jurkat cells, and the mice were randomly assigned to receive control T cells or CART2 or CART5 (1x10 6 ) on day 7. The mice were imaged weekly using an IVIS Xenogen Spectrum and analyzed using Livinglmage software. CART2 C3043 and CART5C3054 were the most effective ( Figure 12 ).

[0513] CART2 and CART5 were shown to recognize normal T cells (autologous and allogeneic) and kill them ( Figure 16 ).

[0514] It was also shown that removal of the CAR target protected normal T cells from CART killing ( Figure 17 ). CD5 KO but not WT normal T cells were resistant to CART5 killing. Normal resting T cells were recognized and killed by CART2 ( Figure 17 , top) and CART5 ( Figure 17 , bottom). Efficient KO of CD2 or CD5 from normal T cells using CRISPR-Cas9 resulted in resistance to CART2 or CART5 killing, respectively ( Figure 17 ).

[0515] CMV-specific T cells were present in the CD2KO and CD5KO normal T cell products ( Figure 18 ). CD2- and CD5 KO normal T cells maintained the ability to recognize CMV peptides and produce cytokines ( Figure 18; 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).

[0516] Example 5: Anti-CD7 CAR T Cells (CART7) and CD7 Knockout (KO) Normal T Cells

[0517] Disclosed herein are two-pronged immunotherapy methods that include anti-CD7 CAR T cells (CART7) and CD7 knockout (KO) normal T cells. CART7 destroys T cell lymphoma (e.g., T-NHL) or T cell leukemia cells, but also kills normal T cells. Infusion of CD7 KO normal T cells provides CART-resistant T cell immunity until CART7 cells are depleted, in some cases by using a suicide gene (e.g., iCasp9).

[0518] Guide RNAs were designed to knockout the CD7 gene using the CRISPR / Cas9 system. CD7 of 79% of the T cell population was effectively knocked out ( Figure 25 ). Six anti-CD7 CARs were generated ( Figure 25 ).

[0519] Example 6: Bispecific CAR T Cells

[0520] Two lentiviral constructs were generated that included CAR5 (C3054) and CAR2 (C3043) linked by a P2A sequence ( Figure 19 ). Gene expression was driven by the EF1α promoter. The CAR5 construct had a 4-1BB co-stimulatory domain and a CD3ζ signaling domain. Effective knockout of CD2 and CD5 in normal T cells was demonstrated ( Figures 20A-20B ).

[0521] Example 6: CD5 KO Enhances CART Immunotherapy

[0522] It was shown that CD5 KO CART5 was more effective than CD5+ CART5 in vivo. CD5 KO increased the anti-tumor efficacy of CART5 ( Figure 21 ). In a Jurkat T-ALL xenograft implantation model using NSG mice, compared to WT CART5, CD5 KO CART5 (2x10 6 cells / mouse) led to complete long-term responses and longer survival ( Figure 21 ).

[0523] CD5 KO CART19 was also more effective than CD5+ CART19 in vivo. CD5 KO increased the anti-tumor efficacy of CART19 ( Figure 22)。In the NALM6 B-ALL xenograft model, compared to WT CART 19, CD5 KO CART19 showed significantly higher tumor control ( Figure 22 )。

[0524] 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 ( Figures 23A-23B )。

[0525] CART5 also targeted 100% of CLL and MCL. Cytotoxicity assays were performed and it was demonstrated that CART5 cells could recognize and kill CD5+ MCL cell lines (Jeko-1 and Mino) ( Figure 24 )。

[0526] These data indicate that knocking out CD5 enhances CART therapy when treated with anti-CD5 CAR or unexpectedly when treated with different CAR T cells (such as CD19 CART cells).

[0527] Other Embodiments

[0528] In the description of the list of elements in any definition of a variable herein, the recitation of the variable includes defining the variable as any single element or a combination (or sub-combination) of the listed elements. The description of the embodiments herein includes the embodiments as any single embodiment or a combination with any other embodiment or a part thereof.

[0529] The disclosure of each patent, patent application, and publication cited herein is incorporated herein by reference in its entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention can be designed by other persons skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations. Sequence Listing <110> The Trustees of the University of Pennsylvania M. Ruella S. Gill C.H. June A.D. Posey D.J. Powell <120> Use of CD2 / 5 / 7 Knockout Anti-CD2 / 5 / 7 Chimeric Antigen Receptor T Cells Against T-Cell Lymphoma and Leukemia <130> 046483-7224WO1(02147) <150> 62 / 782,131 <151> 2018-12-19 <160> 101 <170> PatentIn version 3.5 <210> 1 <211> 747 <212> DNA <213> Artificial sequence <220> <223> CD2-MEDI507H2L-3028 CAR <400> 1 ggatcccaag tccaactggt gcaatcaggc gcagaagtcc aacgaccggg ggccagtgtt 60 aaagtgtctt gtaaagcctc cgggtacatt tttactgagt actatatgta ctgggtcaga 120 caggccccag ggcaaggttt ggaacttgtc ggacgcatag atcccgaaga cggttctata 180 gattacgttg agaagttcaa aaagaaagtc acacttactg cggacacatc tagtagcacc 240 gcatatatgg aactgagcag tctcacctca gacgacaccg cagtgtacta ttgcgctcgc 300 ggaaagttta actataggtt cgcgtactgg ggacagggga cactggtgac tgttagcagc 360 ggtggcggag ggagcggcgg tggaggaagc ggaggcggag gttccgacgt tgtgatgacg 420 caaagtcccc cgtcactcct tgttactctc ggccagccag cgtctatctc ttgccggtca 480 agccagagct tgctccactc tagtggtaac acgtatttga actggttgct gcaaaggcct 540 ggacaatctc ctcagcccct gatctatttg gttagcaaac tggaaagtgg tgttccagac 600 agattttcag ggtctggatc aggcactgat ttcactctga agatctccgg ggtagaggcc 660 gaggacgtgg gagtctatta ctgcatgcag tttactcact atccttatac ctttggtcaa 720 gggacgaaac tggagatcaa atccgga 747 <210> 2 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> CD2-OKT11H2L-3029 <400> 2 ggatcccaag ttcagcttca gcaaccaggt gctgaattgg tccgccctgg aactagcgtt 60 aaactgtctt gtaaggcatc cggttatacg tttacaagtt attggatgca ctggattaag 120 caaaggcccg aacaaggcct tgaatggatt gggagaattg atccctacga tagcgagaca 180 cactacaatg aaaaatttaa agataaggcc atcctcagcg tagataagag cagttctacc 240 gcatacatac agctctcaag cctgacgtca gatgactcag ccgtttatta ttgctcaagg 300 cgggacgcta aatacgacgg ctatgcgctt gactactggg gacaaggcac cactttgaca 360 gtctccagtg gtggcggagg gagcggcggt ggaggaagcg gaggcggagg ttccgatata 420 gttatgacgc aagcagcacc ctctgtacct gtgacaccgg gtgaatccgt tagtatctca 480 tgccgctctt ctaaaaccct cttgcattct aacggcaata catatttgta ttggttcctt 540 caacgaccag gacaatcacc gcaagtgctt atttatagga tgtctaactt ggctagtggg 600 gtgccaaata ggttcagtgg gtctggatct gagacaactt tcacgttgag aataagtagg 660 gtggaagctg aagacgtcgg tatatactac tgtatgcagc atttggagta cccttacact 720 ttcgggggag gtactaagct cgaaattaaa tccgga 756 <210> 3 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> CD2-OKT11L2H-3030 CAR <400> 3 ggatccgata tagttatgac gcaagcagca ccctctgtac ctgtgacacc gggtgaatcc 60 gttagtatct catgccgctc ttctaaaacc ctcttgcatt ctaacggcaa tacatatttg 120 tattggttcc ttcaacgacc aggacaatca ccgcaagtgc ttatttatag gatgtctaac 180 ttggctagtg gggtgccaaa taggttcagt gggtctggat ctgagacaac tttcacgttg 240 agaataagta gggtggaagc tgaagacgtc ggtatatact actgtatgca gcatttggag 300 tacccttaca ctttcggggg aggtactaag ctcgaaatta aaggtggcgg agggagcggc 360 ggtggaggaa gcggaggcgg aggttcccaa gttcagcttc agcaaccagg tgctgaattg 420 gtccgccctg gaactagcgt taaactgtct tgtaaggcat ccggttatac gtttacaagt 480 tattggatgc actggattaa gcaaaggccc gaacaaggcc ttgaatggat tgggagaatt 540 gatccctacg atagcgagac acactacaat gaaaaattta aagataaggc catcctcagc 600 gtagataaga gcagttctac cgcatacata cagctctcaa gcctgacgtc agatgactca 660 gccgtttatt attgctcaag gcgggacgct aaatacgacg gctatgcgct tgactactgg 720 ggacaaggca ccactttgac agtctccagt tccgga 756 <210> 4 <211> 753 <212> DNA <213> Artificial Sequence <220> <223> CD2-T11-2-H2L-3031 CAR <400> 4 ggatcccaag ttcaattgca gcaaccgggt gccgagttgg taaggcccgg tgcgtcagtc 60 ggatcccaag ttcaattgca gcaaccgggt gccgagttgg taaggcccgg tgcgtcagtc 60 aaacttagtt gtaaagctag tgggtacact tttactacgt tctggatgaa ttgggtgaag 120 aaacttagtt gtaaagctag tgggtacact tttactacgt tctggatgaa ttgggtgaag 120 caacgaccag gccaaggtct ggaatggatc ggcatgattg acccgtctga ctcagaagct 180 caacgaccag gccaaggtct ggaatggatc ggcatgattg acccgtctga ctcagaagct 180 cattacaacc agatgttcaa ggacaaggcg actctgactg ttgataaaag ctcaagcacc 240 cattacaacc agatgttcaa ggacaaggcg actctgactg ttgataaaag ctcaagcacc 240 gcctacatgc agctcagtag cctcacatcc gaggattccg cagtgtacta ttgcgcgagg 300 gcctacatgc agctcagtag cctcacatcc gaggattccg cagtgtacta ttgcgcgagg 300 ggacgagggt atgatgacgg cgatgcgatg gactattggg gacaggggac cagcgtaaca 360 ggacgagggt atgatgacgg cgatgcgatg gactattggg gacaggggac cagcgtaaca 360 gtcagtagtg gtggcggagg gagcggcggt ggaggaagcg gaggcggagg ttccgatata 420 gtcagtagtg gtggcggagg gagcggcggt ggaggaagcg gaggcggagg ttccgatata 420 gttatgaccc agtctcccgc ctctctggcc gttagcttgg gacaacgcgc taccatctct 480 gttatgaccc agtctcccgc ctctctggcc gttagcttgg gacaacgcgc taccatctct 480 taccgagcgt ctaagtccgt cagtacaagc ggttatagtt acatgcactg gaaccagcaa 540 taccgagcgt ctaagtccgt cagtacaagc ggttatagtt acatgcactg gaaccagcaa 540 aagcccggac aacctccgag actcctgatt tatttggtct ctaaccttga gtcaggtgtc 600 aagcccggac aacctccgag actcctgatt tatttggtct ctaaccttga gtcaggtgtc 600 ccagccagat tctccggctc tggaagcggc actgacttta cattgaacat tcaccccgtg 660 ccagccagat tctccggctc tggaagcggc actgacttta cattgaacat tcaccccgtg 660 gaggaggaag acgctgctac ctactattgc atgcaattca cgcactatcc ctacacattc 720 gaggaggaag acgctgctac ctactattgc atgcaattca cgcactatcc ctacacattc 720 ggggggggca cgaaattgga aatcaaatcc gga 753 <210> 5 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> CD2-TS2-18.1.1-H2L-3032 CAR <400> 5 ggatccgagg ttcagcttga ggagagtggg ggaggtttgg taatgccagg tgggtctttg 60 aaactcagtt gcgcggcgtc aggcttcgca ttttcctcct acgatatgtc ctgggtcaga 120 cagacacccg agaagcggct ggaatgggtc gcttacattt ccgggggagg attcacgtac 180 tacccggata cagtaaaggg gagatttact ctgagccggg acaacgctaa gaataccctc 240 tatctccaga tgtcctcttt gaagagtgaa gacacagcga tgtattactg tgcgagacaa 300 ggggccaatt gggagctggt ttactggggc caggggacga cattgacggt ttctagcggt 360 ggcggaggga gcggcggtgg aggaagcgga ggcggaggtt ccgacattgt aatgacacaa 420 tcacctgcta cacttagcgt gactccaggt gatcgggtat tcctgagctg ccgcgcatca 480 caaagtatat ccgacttcct gcactggtat cagcagaaat ctcacgaaag tcccaggctg 540 ctgattaaat acgcttccca gagtattagt ggtatcccct cacgattttc tggcagcggg 600 agcggtagtg acttcactct ttctataaac tccgtcgagc cagaagacgt gggggtgtat 660 ctttgccaaa atggacacaa ttttccacca acctttggtg ggggcaccaa actcgaaata 720 aagtccgga 729 <210> 6 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> CD2-TS2-18.1.1-L2H-3033 CAR <400> 6 ggatccgaca ttgtaatgac acaatcacct gctacactta gcgtgactcc aggtgatcgg 60 gtattcctga gctgccgcgc atcacaaagt atatccgact tcctgcactg gtatcagcag 120 aaatctcacg aaagtcccag gctgctgatt aaatacgctt cccagagtat tagtggtatc 180 ccctcacgat tttctggcag cgggagcggt agtgacttca ctctttctat aaactccgtc 240 gagccagaag acgtgggggt gtatctttgc caaaatggac acaattttcc accaaccttt 300 ggtgggggca ccaaactcga aataaagggt ggcggaggga gcggcggtgg aggaagcgga 360 ggcggaggtt ccgaggttca gcttgaggag agtgggggag gtttggtaat gccaggtggg 420 tctttgaaac tcagttgcgc ggcgtcaggc ttcgcatttt cctcctacga tatgtcctgg 480 gtcagacaga cacccgagaa gcggctggaa tgggtcgctt acatttccgg gggaggattc 540 acgtactacc cggatacagt aaaggggaga tttactctga gccgggacaa cgctaagaat 600 accctctatc tccagatgtc ctctttgaag agtgaagaca cagcgatgta ttactgtgcg 660 agacaagggg ccaattggga gctggtttac tggggccagg ggacgacatt gacggtttct 720 agctccgga 729 <210> 7 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> CD2-MEDI507L2H-3043 CAR <400> 7 ggatccgacg ttgtgatgac gcaaagtccc ccgtcactcc ttgttactct cggccagcca 60 gcgtctatct cttgccggtc aagccagagc ttgctccact ctagtggtaa cacgtatttg 120 aactggttgc tgcaaaggcc tggacaatct cctcagcccc tgatctattt ggttagcaaa 180 ctggaaagtg gtgttccaga cagattttca gggtctggat caggcactga tttcactctg 240 aagatctccg gggtagaggc cgaggacgtg ggagtctatt actgcatgca gtttactcac 300 tatccttata cctttggtca agggacgaaa ctggagatca aaggtggcgg agggagcggc 360 ggtggaggaa gcggaggcgg aggttcccaa gtccaactgg tgcaatcagg cgcagaagtc 420 caacgaccgg gggccagtgt taaagtgtct tgtaaagcct ccgggtacat ttttactgag 480 tactatatgt actgggtcag acaggcccca gggcaaggtt tggaacttgt cggacgcata 540 gatcccgaag acggttctat agattacgtt gagaagttca aaaagaaagt cacacttact 600 gcggacacat ctagtagcac cgcatatatg gaactgagca gtctcacctc agacgacacc 660 gcagtgtact attgcgctcg cggaaagttt aactataggt tcgcgtactg gggacagggg 720 acactggtga ctgttagcag ctccgga 747 <210> 8 <211> 738 <212> DNA <213> Artificial Sequence <220> <223> CD5-17L2H-3045 CAR <400> 8 ggatccaaca ttgtactgac gcaaagcccc tcatctttgt ctgagtcact cggcggcaaa 60 gtaaccatca catgcaaggc cagtcaagac atcaataaat atattgcttg gtatcagtat 120 aaacccggca aggggccgcg actgctgatt cactacacga gtaccttgca accgggcatt 180 ccgagccgat ttagtggcag tggctcaggt cgcgattact cattctcaat aagtaatctc 240 gaaccggaag acatagctac ttattattgc ttgcagtacg ataatttgtg gaccttcggg 300 ggtggtacaa agttggaaat aaagggtggc ggagggagcg gcggtggagg aagcggaggc 360 ggaggttccg aggtccaact cgtagaatca ggtcccggat tggtgcaacc atcccagagc 420 ctctctatta catgcacggt ctctggattt agtctgacca attacgatgt gcattgggtg 480 cgccagtctc ccggcaaggg gttggaatgg cttggcgtta tatggaacta cggaaataca 540 gactataacg ccgcgtttat ctctcggctg agtatacgga aagacagtag taaatcccag 600 gtctttttta cgatgtcatc cctgcaaacg ccagataccg caatatatta ctgcgccagg 660 aaccacggtg atggttatta taattggtac ttcgatgtgt ggggtactgg cactacagtc 720 acagtatctt catctaga 738 <210> 9 <211> 741 <212> DNA <213> Artificial Sequence <220> <223> CD5-9H2L-3048 CAR <400> 9 ggatcccagg tccagctgaa agaaagcggt ccagagctgg aaaaacccgg tgcgagcgtc 60 aaaatatcat gtaaagcaag cgggtattca ttcaccgcgt actctatgaa ctgggttaag 120 caaaacaacg gtatgtcctt ggagtggata gggtctatcg acccgtatta tggggacaca 180 aaatacgcgc agaaattcaa ggggaaggcc accctgaccg tagataaagc tagttctact 240 gcgtacttgc aactgaaaag cctcacttct gaggactctg ccgtctacta ctgtgctcgg 300 cgaatgataa cgacggggga ctggtatttc gatgtttggg gtacagggac tacggtgact 360 gtcagtagcg gtggcggagg gagcggcggt ggaggaagcg gaggcggagg ttcccatatc 420 gtcttgactc aatcacctag ttctttgtct gcgtcccttg gcgaccgagt caccatatct 480 tgcagagcgt cacaggacat ttcaacgtac ctcaactggt atcagcaaaa accggacggg 540 actgtcaagc tcttgatctt ctacacttcc agactccacg ccggggtgcc aagcagattt 600 agtggctctg gcagcgggac acaccatagt cttacaatca gcaatcttga gcaagaagac 660 atagccacgt atttctgcca gcaaggtaac tcacttccgt tcacgtttgg tagtggcacc 720 aaactggaga taaaatccgg a 741 <210> 10 <211> 741 <212> DNA <213> Artificial Sequence <220> <223> CD5-9L2H-3049 CAR <400> 10 ggatcccata tcgtcttgac tcaatcacct agttctttgt ctgcgtccct tggcgaccga 60 gtcaccatat cttgcagagc gtcacaggac atttcaacgt acctcaactg gtatcagcaa 120 aaaccggacg ggactgtcaa gctcttgatc ttctacactt ccagactcca cgccggggtg 180 ccaagcagat ttagtggctc tggcagcggg acacaccata gtcttacaat cagcaatctt 240 gagcaagaag acatagccac gtatttctgc cagcaaggta actcacttcc gttcacgttt 300 ggtagtggca ccaaactgga gataaaaggt ggcggaggga gcggcggtgg aggaagcgga 360 ggcggaggtt cccaggtcca gctgaaagaa agcggtccag agctggaaaa acccggtgcg 420 agcgtcaaaa tatcatgtaa agcaagcggg tattcattca ccgcgtactc tatgaactgg 480 gttaagcaaa acaacggtat gtccttggag tggatagggt ctatcgaccc gtattatggg 540 gacacaaaat acgcgcagaa attcaagggg aaggccaccc tgaccgtaga taaagctagt 600 tctactgcgt acttgcaact gaaaagcctc acttctgagg actctgccgt ctactactgt 660 gctcggcgaa tgataacgac gggggactgg tatttcgatg tttggggtac agggactacg 720 gtgactgtca gtagctccgg a 741 <210> 11 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> CD5-34H2L-3052 CAR <400> 11 ggatccgagg ttaaactcgt ggagagcggt gccgaactcg tccgaagtgg tgcttccgtt 60 aaactcagtt gtgccgcgtc aggatttaac ataaaagatt actacattca ctgggtcaaa 120 cagcgcccgg agcaggggct tgaatggatc gggtggattg atcctgaaaa cgggcgcacc 180 gaatatgctc ccaagttcca gggcaaagct actatgaccg ctgacacctc tagtaacact 240 gcctacctgc agttgagctc tcttacgtct gaggataccg ctgtgtacta ctgtaataac 300 ggaaattatg tacgacacta ttacttcgac tactgggggc agggcactac tgtgactgta 360 tctagcggtg gcggagggag cggcggtgga ggaagcggag gcggaggttc cgattggctc 420 acacaatccc ctgcaatcct gagtgcatct ccaggcgaga aagtaactat gacttgcaga 480 gctataagct ctgtgtccta catgcactgg tatcagcaga agccaggttc ttccccgaag 540 ccgtggatat atgctacaag caatttggca tccggtgttc ccgcccggtt tagtggctcc 600 ggttctggga caagttactc cctcacgatc agcagggttg aagccgagga cgctgccact 660 tactattgcc aacagtggtc aagtaacccc aggactttcg ggggaggaac taaacttgaa 720 atcaaatcta ga 732 <210> 12 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> CD5-34L2H-3053 CAR <400> 12 ggatccgatt ggctcacaca atcccctgca atcctgagtg catctccagg cgagaaagta 60 actatgactt gcagagctat aagctctgtg tcctacatgc actggtatca gcagaagcca 120 ggttcttccc cgaagccgtg gatatatgct acaagcaatt tggcatccgg tgttcccgcc 180 cggtttagtg gctccggttc tgggacaagt tactccctca cgatcagcag ggttgaagcc 240 gaggacgctg ccacttacta ttgccaacag tggtcaagta accccaggac tttcggggga 300 ggaactaaac ttgaaatcaa aggtggcgga gggagcggcg gtggaggaag cggaggcgga 360 ggttccgagg ttaaactcgt ggagagcggt gccgaactcg tccgaagtgg tgcttccgtt 420 aaactcagtt gtgccgcgtc aggatttaac ataaaagatt actacattca ctgggtcaaa 480 cagcgcccgg agcaggggct tgaatggatc gggtggattg atcctgaaaa cgggcgcacc 540 gaatatgctc ccaagttcca gggcaaagct actatgaccg ctgacacctc tagtaacact 600 gcctacctgc agttgagctc tcttacgtct gaggataccg ctgtgtacta ctgtaataac 660 ggaaattatg tacgacacta ttacttcgac tactgggggc agggcactac tgtgactgta 720 tctagctcta ga 732 <210> 13 <211> 738 <212> DNA <213> Artificial Sequence <220> <223> CD5-17H2L-3054 CAR <400> 13 ggatccgagg tccaactcgt agaatcaggt cccggattgg tgcaaccatc ccagagcctc 60 tctattacat gcacggtctc tggatttagt ctgaccaatt acgatgtgca ttgggtgcgc 120 cagtctcccg gcaaggggtt ggaatggctt ggcgttatat ggaactacgg aaatacagac 180 tataacgccg cgtttatctc tcggctgagt atacggaaag acagtagtaa atcccaggtc 240 ttttttacga tgtcatccct gcaaacgcca gataccgcaa tatattactg cgccaggaac 300 cacggtgatg gttattataa ttggtacttc gatgtgtggg gtactggcac tacagtcaca 360 gtatcttcag gtggcggagg gagcggcggt ggaggaagcg gaggcggagg ttccaacatt 420 gtactgacgc aaagcccctc atctttgtct gagtcactcg gcggcaaagt aaccatcaca 480 tgcaaggcca gtcaagacat caataaatat attgcttggt atcagtataa acccggcaag 540 gggccgcgac tgctgattca ctacacgagt accttgcaac cgggcattcc gagccgattt 600 agtggcagtg gctcaggtcg cgattactca ttctcaataa gtaatctcga accggaagac 660 atagctactt attattgctt gcagtacgat aatttgtgga ccttcggggg tggtacaaag 720 ttggaaataa agtctaga 738 <210> 14 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> CD8 Transmembrane Domain <400> 14 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gc 72 <210> 15 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> CD8 Transmembrane Domain <400> 15 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 16 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> CD8 Hinge Domain <400> 16 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 17 <211> 45 <212> PRT <213> Artificial sequence <220> <223> CD8 hinge domain <400> 17 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 18 <211> 126 <212> DNA <213> Artificial sequence <220> <223> 4-1BB <400> 18 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 19 <211> 336 <212> DNA <213> Artificial sequence <220> <223> CD3-zeta <400> 19 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 20 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> 4-1BB <400> 20 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 21 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3-zeta <400> 21 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 22 <211> 20 <212> DNA <213> Artificial sequence <220> <223> gRNA <400> 22 acagctgaca ggctcgacac 20 <210> 23 <211> 20 <212> DNA <213> Artificial sequence <220> <223> gRNA <400> 23 cggctcagct ggtatgaccc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> gRNA <400> 24 ggagcaggtg atgttgacgg 20 <210> 25 <211> 493 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507H2L-3028 CAR <400> 25 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala 20 25 30 Glu Val Gln Arg Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Ile Phe Thr Glu Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro 50 55 60 Gly Gln Gly Leu Glu Leu Val Gly Arg Ile Asp Pro Glu Asp Gly Ser 65 70 75 80 Ile Asp Tyr Val Glu Lys Phe Lys Lys Lys Val Thr Leu Thr Ala Asp 85 90 95 Thr Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Asp 100 105 110 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Phe Asn Tyr Arg Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met 145 150 155 160 Thr Gln Ser Pro Pro Ser Leu Leu Val Thr Leu Gly Gln Pro Ala Ser 165 170 175 Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Ser Gly Asn Thr 180 185 190 Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Gln Pro Leu 195 200 205 Ile Tyr Leu Val Ser Lys Leu Glu Ser Gly Val Pro Asp Arg Phe Ser 210 215 220 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Gly Val Glu 225 230 235 240 Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Phe Thr His Tyr Pro 245 250 255 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Gly Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 305 310 315 320 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 325 330 335 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 340 345 350 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 355 360 365 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 370 375 380 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln 385 390 395 400 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 405 410 415 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 420 425 430 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 435 440 445 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 450 455 460 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 465 470 475 480 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 26 <211> 493 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507L2H-3043 CAR <400> 26 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Asp Val Val Met Thr Gln Ser Pro Pro 20 25 30 Ser Leu Leu Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser 35 40 45 Ser Gln Ser Leu Leu His Ser Ser Gly Asn Thr Tyr Leu Asn Trp Leu 50 55 60 Leu Gln Arg Pro Gly Gln Ser Pro Gln Pro Leu Ile Tyr Leu Val Ser 65 70 75 80 Lys Leu Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 85 90 95 Thr Asp Phe Thr Leu Lys Ile Ser Gly Val Glu Ala Glu Asp Val Gly 100 105 110 Val Tyr Tyr Cys Met Gln Phe Thr His Tyr Pro Tyr Thr Phe Gly Gln 115 120 125 Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 145 150 155 160 Val Gln Arg Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 165 170 175 Tyr Ile Phe Thr Glu Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly 180 185 190 Gln Gly Leu Glu Leu Val Gly Arg Ile Asp Pro Glu Asp Gly Ser Ile 195 200 205 Asp Tyr Val Glu Lys Phe Lys Lys Lys Val Thr Leu Thr Ala Asp Thr 210 215 220 Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Asp Asp 225 230 235 240 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Phe Asn Tyr Arg Phe Ala 245 250 255 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 305 310 315 320 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 325 330 335 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 340 345 350 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 355 360 365 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 370 375 380 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln 385 390 395 400 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 405 410 415 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 420 425 430 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 435 440 445 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 450 455 460 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 465 470 475 480 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 27 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507H2L-3028 scFv <400> 27 Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Gln Arg Pro 1 5 10 15 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr 20 25 30 Glu Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Leu Val Gly Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu 50 55 60 Lys Phe Lys Lys Lys Val Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr 65 70 75 80 Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Lys Phe Asn Tyr Arg Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser Pro Pro 130 135 140 Ser Leu Leu Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser 145 150 155 160 Ser Gln Ser Leu Leu His Ser Ser Gly Asn Thr Tyr Leu Asn Trp Leu 165 170 175 Leu Gln Arg Pro Gly Gln Ser Pro Gln Pro Leu Ile Tyr Leu Val Ser 180 185 190 Lys Leu Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Lys Ile Ser Gly Val Glu Ala Glu Asp Val Gly 210 215 220 Val Tyr Tyr Cys Met Gln Phe Thr His Tyr Pro Tyr Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Ser Gly 245 <210> 28 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507L2H-3043 scFv <400> 28 Gly Ser Asp Val Val Met Thr Gln Ser Pro Pro Ser Leu Leu Val Thr 1 5 10 15 Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 20 25 30 His Ser Ser Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly 35 40 45 Gln Ser Pro Gln Pro Leu Ile Tyr Leu Val Ser Lys Leu Glu Ser Gly 50 55 60 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 65 70 75 80 Lys Ile Ser Gly Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met 85 90 95 Gln Phe Thr His Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 100 105 110 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Gln Arg Pro Gly 130 135 140 Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Glu 145 150 155 160 Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Leu 165 170 175 Val Gly Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu Lys 180 185 190 Phe Lys Lys Lys Val Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala 195 200 205 Tyr Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr 210 215 220 Cys Ala Arg Gly Lys Phe Asn Tyr Arg Phe Ala Tyr Trp Gly Gln Gly 225 230 235 240 Thr Leu Val Thr Val Ser Ser Ser Gly 245 <210> 29 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507 VH <400> 29 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Gln Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Glu Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Leu Val 35 40 45 Gly Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu Lys Phe 50 55 60 Lys Lys Lys Val Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Lys Phe Asn Tyr Arg Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 30 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507 VL <400> 30 Asp Val Val Met Thr Gln Ser Pro Pro Ser Leu Leu Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Ser Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Pro Leu Ile Tyr Leu Val Ser Lys Leu Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Gly Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Phe 85 90 95 Thr His Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 31 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507 HCDR1 <400> 31 Glu Tyr Tyr Met Tyr 1 5 <210> 32 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507 HCDR2 <400> 32 Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu Lys Phe Lys 1 5 10 15 Lys <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507 HCDR3 <400> 33 Gly Lys Phe Asn Tyr Arg Phe Ala Tyr 1 5 <210> 34 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507 LCDR1 <400> 34 Arg Ser Ser Gln Ser Leu Leu His Ser Ser Gly Asn Thr Tyr Leu Asn 1 5 10 15 <210> 35 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507 LCDR2 <400> 35 Leu Val Ser Lys Leu Glu Ser 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CD2-MEDI507 LCDR3 <400> 36 Met Gln Phe Thr His Tyr Pro Tyr Thr 1 5 <210> 37 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> CD2-OKT11H2L-3029 CAR <400> 37 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala 20 25 30 Glu Leu Val Arg Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro 50 55 60 Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Tyr Asp Ser Glu 65 70 75 80 Thr His Tyr Asn Glu Lys Phe Lys Asp Lys Ala Ile Leu Ser Val Asp 85 90 95 Lys Ser Ser Ser Thr Ala Tyr Ile Gln Leu Ser Ser Leu Thr Ser Asp 100 105 110 Asp Ser Ala Val Tyr Tyr Cys Ser Arg Arg Asp Ala Lys Tyr Asp Gly 115 120 125 Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 145 150 155 160 Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly Glu 165 170 175 Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Thr Leu Leu His Ser Asn 180 185 190 Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro 195 200 205 Gln Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asn 210 215 220 Arg Phe Ser Gly Ser Gly Ser Glu Thr Thr Phe Thr Leu Arg Ile Ser 225 230 235 240 Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln His Leu 245 250 255 Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser 260 265 270 Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 275 280 285 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 290 295 300 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 305 310 315 320 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 325 330 335 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 340 345 350 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 355 360 365 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 370 375 380 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 385 390 395 400 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 405 410 415 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 420 425 430 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 435 440 445 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 450 455 460 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 465 470 475 480 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 495 <210> 38 <211> 496 <212> PRT <213> Artificial Sequence <220> <223> CD2-OKT11L2H-3030 CAR <400> 38 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Asp Ile Val Met Thr Gln Ala Ala Pro 20 25 30 Ser Val Pro Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser 35 40 45 Ser Lys Thr Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe 50 55 60 Leu Gln Arg Pro Gly Gln Ser Pro Gln Val Leu Ile Tyr Arg Met Ser 65 70 75 80 Asn Leu Ala Ser Gly Val Pro Asn Arg Phe Ser Gly Ser Gly Ser Glu 85 90 95 Thr Thr Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly 100 105 110 Ile Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Tyr Thr Phe Gly Gly 115 120 125 Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu 145 150 155 160 Leu Val Arg Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly 165 170 175 Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro Glu 180 185 190 Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr 195 200 205 His Tyr Asn Glu Lys Phe Lys Asp Lys Ala Ile Leu Ser Val Asp Lys 210 215 220 Ser Ser Ser Thr Ala Tyr Ile Gln Leu Ser Ser Leu Thr Ser Asp Asp 225 230 235 240 Ser Ala Val Tyr Tyr Cys Ser Arg Arg Asp Ala Lys Tyr Asp Gly Tyr 245 250 255 Ala Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ser 260 265 270 Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 275 280 285 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 290 295 300 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 305 310 315 320 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 325 330 335 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 340 345 350 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 355 360 365 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 370 375 380 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 385 390 395 400 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 405 410 415 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 420 425 430 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 435 440 445 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 450 455 460 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 465 470 475 480 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 495 <210> 39 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> CD2-OKT11H2L-3029 scFv <400> 39 Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro 1 5 10 15 Gly Thr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Ser Tyr Trp Met His Trp Ile Lys Gln Arg Pro Glu Gln Gly Leu Glu 35 40 45 Trp Ile Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Glu 50 55 60 Lys Phe Lys Asp Lys Ala Ile Leu Ser Val Asp Lys Ser Ser Ser Thr 65 70 75 80 Ala Tyr Ile Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr 85 90 95 Tyr Cys Ser Arg Arg Asp Ala Lys Tyr Asp Gly Tyr Ala Leu Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln 130 135 140 Ala Ala Pro Ser Val Pro Val Thr Pro Gly Glu Ser Val Ser Ile Ser 145 150 155 160 Cys Arg Ser Ser Lys Thr Leu Leu His Ser Asn Gly Asn Thr Tyr Leu 165 170 175 Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Val Leu Ile Tyr 180 185 190 Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asn Arg Phe Ser Gly Ser 195 200 205 Gly Ser Glu Thr Thr Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu 210 215 220 Asp Val Gly Ile Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Tyr Thr 225 230 235 240 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Gly 245 250 <210> 40 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> CD2-OKT11L2H-3030 scFv <400> 40 Gly Ser Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr 1 5 10 15 Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Thr Leu Leu 20 25 30 His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly 35 40 45 Gln Ser Pro Gln Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly 50 55 60 Val Pro Asn Arg Phe Ser Gly Ser Gly Ser Glu Thr Thr Phe Thr Leu 65 70 75 80 Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met 85 90 95 Gln His Leu Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 100 105 110 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly 130 135 140 Thr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser 145 150 155 160 Tyr Trp Met His Trp Ile Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp 165 170 175 Ile Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Glu Lys 180 185 190 Phe Lys Asp Lys Ala Ile Leu Ser Val Asp Lys Ser Ser Ser Thr Ala 195 200 205 Tyr Ile Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr 210 215 220 Cys Ser Arg Arg Asp Ala Lys Tyr Asp Gly Tyr Ala Leu Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ser Gly 245 250 <210> 41 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> CD2-OKT11 VH <400> 41 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Ile Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Lys Ala Ile Leu Ser Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Ile Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Arg Asp Ala Lys Tyr Asp Gly Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 42 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD2-OKT11 VL <400> 42 Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Thr Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asn Arg Phe Ser Gly Ser Gly Ser Glu Thr Thr Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CD2-OKT11 HCDR1 <400> 43 Ser Tyr Trp Met His 1 5 <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CD2-OKT11 HCDR2 <400> 44 Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Glu Lys Phe Lys 1 5 10 15 Asp <210> 45 <211> 12 <212> PRT <213> Artificial sequence <220> <223> CD2-OKT11 HCDR3 <400> 45 Arg Asp Ala Lys Tyr Asp Gly Tyr Ala Leu Asp Tyr 1 5 10 <210> 46 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CD2-OKT11 LCDR1 <400> 46 Arg Ser Ser Lys Thr Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr 1 5 10 15 <210> 47 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CD2-OKT11 LCDR2 <400> 47 Arg Met Ser Asn Leu Ala Ser 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CD2-OKT11 LCDR3 <400> 48 Met Gln His Leu Glu Tyr Pro Tyr Thr 1 5 <210> 49 <211> 495 <212> PRT <213> Artificial sequence <220> <223> CD2-T11-2-H2L-3031 CAR <400> 49 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala 20 25 30 Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Thr Phe Thr Thr Phe Trp Met Asn Trp Val Lys Gln Arg Pro 50 55 60 Gly Gln Gly Leu Glu Trp Ile Gly Met Ile Asp Pro Ser Asp Ser Glu 65 70 75 80 Ala His Tyr Asn Gln Met Phe Lys Asp Lys Ala Thr Leu Thr Val Asp 85 90 95 Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu 100 105 110 Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Arg Gly Tyr Asp Asp Gly 115 120 125 Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 145 150 155 160 Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln 165 170 175 Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly 180 185 190 Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg 195 200 205 Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala Arg 210 215 220 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro 225 230 235 240 Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Met Gln Phe Thr His 245 250 255 Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Gly 260 265 270 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280 285 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 290 295 300 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 305 310 315 320 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 325 330 335 Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 340 345 350 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 355 360 365 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 370 375 380 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln 385 390 395 400 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 405 410 415 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 420 425 430 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 435 440 445 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 450 455 460 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 465 470 475 480 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 495 <210> 50 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> CD2-T11-2-H2L-3031 scFv <400> 50 Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro 1 5 10 15 Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Thr Phe Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu 35 40 45 Trp Ile Gly Met Ile Asp Pro Ser Asp Ser Glu Ala His Tyr Asn Gln 50 55 60 Met Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 65 70 75 80 Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Arg Gly Tyr Asp Asp Gly Asp Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln 130 135 140 Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser 145 150 155 160 Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 165 170 175 Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu 180 185 190 Val Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp 210 215 220 Ala Ala Thr Tyr Tyr Cys Met Gln Phe Thr His Tyr Pro Tyr Thr Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Gly 245 250 <210> 51 <211> 121 <212> PRT <213> Artificial sequence <220> <223> CD2-T11-2-H2L-3031 VH <400> 51 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Phe 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Ala His Tyr Asn Gln Met Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Gly Tyr Asp Asp Gly Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 52 <211> 111 <212> PRT <213> Artificial sequence <220> <223> CD2-T11-2-H2L-3031 VL <400> 52 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Met Gln Phe Thr 85 90 95 His Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 53 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CD2-T11-2 HCDR1 <400> 53 Thr Phe Trp Met Asn 1 5 <210> 54 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CD2-T11-2 HCDR2 <400> 54 Met Ile Asp Pro Ser Asp Ser Glu Ala His Tyr Asn Gln Met Phe Lys 1 5 10 15 Asp <210> 55 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CD2-T11-2 HCDR3 <400> 55 Gly Arg Gly Tyr Asp Asp Gly Asp Ala Met Asp Tyr 1 5 10 <210> 56 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CD2-T11-2 LCDR1 <400> 56 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 57 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CD2-T11-2 LCDR2 <400> 57 Leu Val Ser Asn Leu Glu Ser 1 5 <210> 58 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CD2-T11-2 LCDR3 <400> 58 Met Gln Phe Thr His Tyr Pro Tyr Thr 1 5 <210> 59 <211> 487 <212> PRT <213> Artificial sequence <220> <223> CD2-TS2-18.1.1-H2L-3032 CAR <400> 59 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Glu Val Gln Leu Glu Glu Ser Gly Gly 20 25 30 Gly Leu Val Met Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser 35 40 45 Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Thr Pro 50 55 60 Glu Lys Arg Leu Glu Trp Val Ala Tyr Ile Ser Gly Gly Gly Phe Thr 65 70 75 80 Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn 85 90 95 Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp 100 105 110 Thr Ala Met Tyr Tyr Cys Ala Arg Gln Gly Ala Asn Trp Glu Leu Val 115 120 125 Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr 145 150 155 160 Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val Phe Leu 165 170 175 Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Phe Leu His Trp Tyr Gln 180 185 190 Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln 195 200 205 Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser 210 215 220 Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val 225 230 235 240 Tyr Leu Cys Gln Asn Gly His Asn Phe Pro Pro Thr Phe Gly Gly Gly 245 250 255 Thr Lys Leu Glu Ile Lys Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro 260 265 270 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg 485 <210> 60 <211> 487 <212> PRT <213> Artificial Sequence <220> <223> CD2-TS2-18.1.1-L2H-3033 CAR <400> 60 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala 20 25 30 Thr Leu Ser Val Thr Pro Gly Asp Arg Val Phe Leu Ser Cys Arg Ala 35 40 45 Ser Gln Ser Ile Ser Asp Phe Leu His Trp Tyr Gln Gln Lys Ser His 50 55 60 Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly 65 70 75 80 Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu 85 90 95 Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Leu Cys Gln 100 105 110 Asn Gly His Asn Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Glu Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Met Pro Gly 145 150 155 160 Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser 165 170 175 Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp 180 185 190 Val Ala Tyr Ile Ser Gly Gly Gly Phe Thr Tyr Tyr Pro Asp Thr Val 195 200 205 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 210 215 220 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 225 230 235 240 Ala Arg Gln Gly Ala Asn Trp Glu Leu Val Tyr Trp Gly Gln Gly Thr 245 250 255 Thr Leu Thr Val Ser Ser Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro 260 265 270 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg 485 <210> 61 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> CD2-TS2-18.1.1-H2L-3032 scFv <400> 61 Gly Ser Glu Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Met Pro 1 5 10 15 Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser 20 25 30 Ser Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu 35 40 45 Trp Val Ala Tyr Ile Ser Gly Gly Gly Phe Thr Tyr Tyr Pro Asp Thr 50 55 60 Val Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr 85 90 95 Cys Ala Arg Gln Gly Ala Asn Trp Glu Leu Val Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala Thr 130 135 140 Leu Ser Val Thr Pro Gly Asp Arg Val Phe Leu Ser Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser Asp Phe Leu His Trp Tyr Gln Gln Lys Ser His Glu 165 170 175 Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser 195 200 205 Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Leu Cys Gln Asn 210 215 220 Gly His Asn Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Ser Gly <210> 62 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> CD2-TS2-18.1.1-L2H-3033 scFv <400> 62 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr 1 5 10 15 Pro Gly Asp Arg Val Phe Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30 Asp Phe Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu 35 40 45 Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val 65 70 75 80 Glu Pro Glu Asp Val Gly Val Tyr Leu Cys Gln Asn Gly His Asn Phe 85 90 95 Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 115 120 125 Glu Glu Ser Gly Gly Gly Leu Val Met Pro Gly Gly Ser Leu Lys Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser Trp 145 150 155 160 Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Tyr Ile Ser 165 170 175 Gly Gly Gly Phe Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr 180 185 190 Leu Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser 195 200 205 Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Gly Ala 210 215 220 Asn Trp Glu Leu Val Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 225 230 235 240 Ser Ser Gly <210> 63 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> CD2-TS2-18.1.1 VH <400> 63 Glu Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Met Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Gly Gly Gly Phe Thr Tyr Tyr Pro Asp Thr Val Lys 50 55 60 Gly Arg Phe Thr Leu Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Gln Gly Ala Asn Trp Glu Leu Val Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 64 <211> 107 <212> PRT <213> Artificial sequence <220> <223> CD2-TS2-18.1.1 VL <400> 64 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Phe Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Phe 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Leu Cys Gln Asn Gly His Asn Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 65 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CD2-TS2-18.1.1 HCDR1 <400> 65 Ser Tyr Asp Met Ser 1 5 <210> 66 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CD2-TS2-18.1.1 HCDR2 <400> 66 Tyr Ile Ser Gly Gly Gly Phe Thr Tyr Tyr Pro Asp Thr Val Lys Gly 1 5 10 15 <210> 67 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CD2-TS2-18.1.1 HCDR3 <400> 67 Gln Gly Ala Asn Trp Glu Leu Val Tyr 1 5 <210> 68 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CD2-TS2-18.1.1 LCDR1 <400> 68 Arg Ala Ser Gln Ser Ile Ser Asp Phe Leu His 1 5 10 <210> 69 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CD2-TS2-18.1.1 LCDR2 <400> 69 Tyr Ala Ser Gln Ser Ile Ser 1 5 <210> 70 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CD2-TS2-18.1.1 LCDR3 <400> 70 Gln Asn Gly His Asn Phe Pro Pro Thr 1 5 <210> 71 <211> 494 <212> PRT <213> Artificial Sequence <220> <223> CD5-17L2H-3045 CAR <400> 71 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Asn Ile Val Leu Thr Gln Ser Pro Ser 20 25 30 Ser Leu Ser Glu Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala 35 40 45 Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln Tyr Lys Pro Gly 50 55 60 Lys Gly Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly 65 70 75 80 Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe 85 90 95 Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu 100 105 110 Gln Tyr Asp Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Glu Val Gln Leu Val Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 145 150 155 160 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 165 170 175 Asp Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 180 185 190 Gly Val Ile Trp Asn Tyr Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile 195 200 205 Ser Arg Leu Ser Ile Arg Lys Asp Ser Ser Lys Ser Gln Val Phe Phe 210 215 220 Thr Met Ser Ser Leu Gln Thr Pro Asp Thr Ala Ile Tyr Tyr Cys Ala 225 230 235 240 Arg Asn His Gly Asp Gly Tyr Tyr Asn Trp Tyr Phe Asp Val Trp Gly 245 250 255 Thr Gly Thr Thr Val Thr Val Ser Ser Ser Arg Thr Thr Thr Pro Ala 260 265 270 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 275 280 285 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 305 310 315 320 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 325 330 335 His Met Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Thr Ser Arg Val 370 375 380 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 385 390 395 400 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 405 410 415 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 420 425 430 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 435 440 445 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 450 455 460 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 465 470 475 480 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 72 <211> 494 <212> PRT <213> Artificial Sequence <220> <223> CD5-17H2L-3054 CAR <400> 72 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Glu Val Gln Leu Val Glu Ser Gly Pro 20 25 30 Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser 35 40 45 Gly Phe Ser Leu Thr Asn Tyr Asp Val His Trp Val Arg Gln Ser Pro 50 55 60 Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Asn Tyr Gly Asn Thr 65 70 75 80 Asp Tyr Asn Ala Ala Phe Ile Ser Arg Leu Ser Ile Arg Lys Asp Ser 85 90 95 Ser Lys Ser Gln Val Phe Phe Thr Met Ser Ser Leu Gln Thr Pro Asp 100 105 110 Thr Ala Ile Tyr Tyr Cys Ala Arg Asn His Gly Asp Gly Tyr Tyr Asn 115 120 125 Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn 145 150 155 160 Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Glu Ser Leu Gly Gly 165 170 175 Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr Ile 180 185 190 Ala Trp Tyr Gln Tyr Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile His 195 200 205 Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser 210 215 220 Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu 225 230 235 240 Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr Phe 245 250 255 Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Arg Thr Thr Thr Pro Ala 260 265 270 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 275 280 285 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 305 310 315 320 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 325 330 335 His Met Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Thr Ser Arg Val 370 375 380 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 385 390 395 400 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 405 410 415 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 420 425 430 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 435 440 445 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 450 455 460 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 465 470 475 480 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 73 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> CD5-17L2H-3045 scFv <400> 73 Asn Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Glu Ser Leu Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln Tyr Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 115 120 125 Gly Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr 130 135 140 Val Ser Gly Phe Ser Leu Thr Asn Tyr Asp Val His Trp Val Arg Gln 145 150 155 160 Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Asn Tyr Gly 165 170 175 Asn Thr Asp Tyr Asn Ala Ala Phe Ile Ser Arg Leu Ser Ile Arg Lys 180 185 190 Asp Ser Ser Lys Ser Gln Val Phe Phe Thr Met Ser Ser Leu Gln Thr 195 200 205 Pro Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Asn His Gly Asp Gly Tyr 210 215 220 Tyr Asn Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val 225 230 235 240 Ser Ser <210> 74 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> CD5-17H2L-3054 scFv <400> 74 Glu Val Gln Leu Val Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Asp Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Asn Tyr Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Ser Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Thr Met Ser Ser Leu Gln Thr Pro Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Asn His Gly Asp Gly Tyr Tyr Asn Trp Tyr Phe Asp Val Trp Gly 100 105 110 Thr Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asn Ile Val Leu Thr Gln Ser Pro 130 135 140 Ser Ser Leu Ser Glu Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys 145 150 155 160 Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln Tyr Lys Pro 165 170 175 Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro 180 185 190 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser 195 200 205 Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys 210 215 220 Leu Gln Tyr Asp Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys <210> 75 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> CD5-17 VH <400> 75 Glu Val Gln Leu Val Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Asp Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Asn Tyr Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Ser Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Thr Met Ser Ser Leu Gln Thr Pro Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Asn His Gly Asp Gly Tyr Tyr Asn Trp Tyr Phe Asp Val Trp Gly 100 105 110 Thr Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 76 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> CD5-17 VL <400> 76 Asn Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Glu Ser Leu Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln Tyr Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 77 <211> 491 <212> PRT <213> Artificial Sequence <220> <223> CD5-9H2L-3048 CAR <400> 77 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Gln Val Gln Leu Lys Glu Ser Gly Pro 20 25 30 Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Ser Phe Thr Ala Tyr Ser Met Asn Trp Val Lys Gln Asn Asn 50 55 60 Gly Met Ser Leu Glu Trp Ile Gly Ser Ile Asp Pro Tyr Tyr Gly Asp 65 70 75 80 Thr Lys Tyr Ala Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp 85 90 95 Lys Ala Ser Ser Thr Ala Tyr Leu Gln Leu Lys Ser Leu Thr Ser Glu 100 105 110 Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Met Ile Thr Thr Gly Asp 115 120 125 Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His 145 150 155 160 Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Asp 165 170 175 Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Thr Tyr Leu 180 185 190 Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Phe 195 200 205 Tyr Thr Ser Arg Leu His Ala Gly Val Pro Ser Arg Phe Ser Gly Ser 210 215 220 Gly Ser Gly Thr His His Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu 225 230 235 240 Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Ser Leu Pro Phe Thr 245 250 255 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly Thr Thr Thr Pro 260 265 270 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 305 310 315 320 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375 380 Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr 385 390 395 400 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440 445 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 465 470 475 480 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 78 <211> 491 <212> PRT <213> Artificial Sequence <220> <223> CD5-9L2H-3049 CAR <400> 78 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser His Ile Val Leu Thr Gln Ser Pro Ser 20 25 30 Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala 35 40 45 Ser Gln Asp Ile Ser Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp 50 55 60 Gly Thr Val Lys Leu Leu Ile Phe Tyr Thr Ser Arg Leu His Ala Gly 65 70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His His Ser Leu 85 90 95 Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln 100 105 110 Gln Gly Asn Ser Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu 115 120 125 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Glu Leu Glu Lys Pro Gly 145 150 155 160 Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ala 165 170 175 Tyr Ser Met Asn Trp Val Lys Gln Asn Asn Gly Met Ser Leu Glu Trp 180 185 190 Ile Gly Ser Ile Asp Pro Tyr Tyr Gly Asp Thr Lys Tyr Ala Gln Lys 195 200 205 Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ala Ser Ser Thr Ala 210 215 220 Tyr Leu Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 225 230 235 240 Cys Ala Arg Arg Met Ile Thr Thr Gly Asp Trp Tyr Phe Asp Val Trp 245 250 255 Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ser Gly Thr Thr Thr Pro 260 265 270 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 305 310 315 320 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375 380 Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr 385 390 395 400 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440 445 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 465 470 475 480 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 79 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> CD5-9H2L-3048 scFv <400> 79 Gly Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Glu Leu Glu Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr 20 25 30 Ala Tyr Ser Met Asn Trp Val Lys Gln Asn Asn Gly Met Ser Leu Glu 35 40 45 Trp Ile Gly Ser Ile Asp Pro Tyr Tyr Gly Asp Thr Lys Tyr Ala Gln 50 55 60 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ala Ser Ser Thr 65 70 75 80 Ala Tyr Leu Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Arg Met Ile Thr Thr Gly Asp Trp Tyr Phe Asp Val 100 105 110 Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Ile Val Leu Thr Gln 130 135 140 Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser 145 150 155 160 Cys Arg Ala Ser Gln Asp Ile Ser Thr Tyr Leu Asn Trp Tyr Gln Gln 165 170 175 Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Phe Tyr Thr Ser Arg Leu 180 185 190 His Ala Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr His 195 200 205 His Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr 210 215 220 Phe Cys Gln Gln Gly Asn Ser Leu Pro Phe Thr Phe Gly Ser Gly Thr 225 230 235 240 Lys Leu Glu Ile Lys Ser Gly 245 <210> 80 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> CD5-9L2H-3049 scFv <400> 80 His Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Phe Tyr Thr Ser Arg Leu His Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr His His Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Ser Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Glu 115 120 125 Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Ser Phe Thr Ala Tyr Ser Met Asn Trp Val Lys 145 150 155 160 Gln Asn Asn Gly Met Ser Leu Glu Trp Ile Gly Ser Ile Asp Pro Tyr 165 170 175 Tyr Gly Asp Thr Lys Tyr Ala Gln Lys Phe Lys Gly Lys Ala Thr Leu 180 185 190 Thr Val Asp Lys Ala Ser Ser Thr Ala Tyr Leu Gln Leu Lys Ser Leu 195 200 205 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Met Ile Thr 210 215 220 Thr Gly Asp Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr 225 230 235 240 Val Ser Ser <210> 81 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> CD5-9 VH <400> 81 Gln Val Gln Leu Lys Glu Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ala Tyr 20 25 30 Ser Met Asn Trp Val Lys Gln Asn Asn Gly Met Ser Leu Glu Trp Ile 35 40 45 Gly Ser Ile Asp Pro Tyr Tyr Gly Asp Thr Lys Tyr Ala Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ala Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Met Ile Thr Thr Gly Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Thr Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 82 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CD5-9 VL <400> 82 His Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Phe Tyr Thr Ser Arg Leu His Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr His His Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Ser Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 83 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CD5-9 HCDR1 <400> 83 Ala Tyr Ser Met Asn 1 5 <210> 84 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CD5-9 HCDR2 <400> 84 Ser Ile Asp Pro Tyr Tyr Gly Asp Thr Lys Tyr Ala Gln Lys Phe Lys 1 5 10 15 Gly <210> 85 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CD5-9 HCDR3 <400> 85 Arg Met Ile Thr Thr Gly Asp Trp Tyr Phe Asp Val 1 5 10 <210> 86 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CD5-9 LCDR1 <400> 86 Arg Ala Ser Gln Asp Ile Ser Thr Tyr Leu Asn 1 5 10 <210> 87 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CD5-9 LCDR2 <400> 87 Tyr Thr Ser Arg Leu His Ala 1 5 <210> 88 <211> 9 <212> PRT <213> Synthetic sequence <220> <223> CD5-9 LCDR3 <400> 88 Gln Gln Gly Asn Ser Leu Pro Phe Thr 1 5 <210> 89 <211> 492 <212> PRT <213> Synthetic sequence <220> <223> CD5-34H2L-3052 CAR <400> 89 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Glu Val Lys Leu Val Glu Ser Gly Ala 20 25 30 Glu Leu Val Arg Ser Gly Ala Ser Val Lys Leu Ser Cys Ala Ala Ser 35 40 45 Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His Trp Val Lys Gln Arg Pro 50 55 60 Glu Gln Gly Leu Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Arg 65 70 75 80 Thr Glu Tyr Ala Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala Asp 85 90 95 Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu 100 105 110 Asp Thr Ala Val Tyr Tyr Cys Asn Asn Gly Asn Tyr Val Arg His Tyr 115 120 125 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Trp 145 150 155 160 Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val 165 170 175 Thr Met Thr Cys Arg Ala Ile Ser Ser Val Ser Tyr Met His Trp Tyr 180 185 190 Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser 195 200 205 Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 210 215 220 Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala 225 230 235 240 Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Arg Thr Phe Gly Gly 245 250 255 Gly Thr Lys Leu Glu Ile Lys Ser Arg Thr Thr Thr Pro Ala Pro Arg 260 265 270 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275 280 285 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 290 295 300 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 305 310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys His Met 325 330 335 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 340 345 350 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 355 360 365 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Thr Ser Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 90 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> CD5-34L2H-3053 CAR <400> 90 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gly Ser Asp Trp Leu Thr Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ile 35 40 45 Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 130 135 140 Val Lys Leu Val Glu Ser Gly Ala Glu Leu Val Arg Ser Gly Ala Ser 145 150 155 160 Val Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr 165 170 175 Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly 180 185 190 Trp Ile Asp Pro Glu Asn Gly Arg Thr Glu Tyr Ala Pro Lys Phe Gln 195 200 205 Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu 210 215 220 Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Asn 225 230 235 240 Asn Gly Asn Tyr Val Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Thr Val Thr Val Ser Ser Ser Arg Thr Thr Thr Pro Ala Pro Arg 260 265 270 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275 280 285 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 290 295 300 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 305 310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys His Met 325 330 335 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 340 345 350 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 355 360 365 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Thr Ser Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 91 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> CD5-34H2L-3052 scFv <400> 91 Glu Val Lys Leu Val Glu Ser Gly Ala Glu Leu Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Arg Thr Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Asn Gly Asn Tyr Val Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Trp Leu Thr Gln Ser Pro Ala Ile 130 135 140 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ile 145 150 155 160 Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser 165 170 175 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 180 185 190 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 195 200 205 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 210 215 220 Ser Ser Asn Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 225 230 235 240 <210> 92 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> CD5-34L2H-3053 scFv <400> 92 Asp Trp Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu 1 5 10 15 Lys Val Thr Met Thr Cys Arg Ala Ile Ser Ser Val Ser Tyr Met His 20 25 30 Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala 35 40 45 Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 50 55 60 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp 65 70 75 80 Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Arg Thr Phe 85 90 95 Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Val Glu Ser Gly 115 120 125 Ala Glu Leu Val Arg Ser Gly Ala Ser Val Lys Leu Ser Cys Ala Ala 130 135 140 Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His Trp Val Lys Gln Arg 145 150 155 160 Pro Glu Gln Gly Leu Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly 165 170 175 Arg Thr Glu Tyr Ala Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala 180 185 190 Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Asn Gly Asn Tyr Val Arg His 210 215 220 Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 225 230 235 240 <210> 93 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> CD5-34 VH <400> 93 Glu Val Lys Leu Val Glu Ser Gly Ala Glu Leu Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Arg Thr Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Asn Gly Asn Tyr Val Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 94 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CD5-34 VL <400> 94 Asp Trp Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu 1 5 10 15 Lys Val Thr Met Thr Cys Arg Ala Ile Ser Ser Val Ser Tyr Met His 20 25 30 Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala 35 40 45 Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 50 55 60 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp 65 70 75 80 Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Arg Thr Phe 85 90 95 Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Arg 100 105 <210> 95 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CD5-34 HCDR1 <400> 95 Asp Tyr Tyr Ile His 1 5 <210> 96 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CD5-34 HCDR2 <400> 96 Trp Ile Asp Pro Glu Asn Gly Arg Thr Glu Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly <210> 97 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CD5-34 HCDR3 <400> 97 Gly Asn Tyr Val Arg His Tyr Tyr Phe Asp Tyr 1 5 10 <210> 98 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CD5-34 LCDR1 <400> 98 Arg Ala Ile Ser Ser Val Ser Tyr Met His 1 5 10 <210> 99 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CD5-34 LCDR2 <400> 99 Ala Thr Ser Asn Leu Ala Ser 1 5 <210> 100 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CD5-34 LCDR3 <400> 100 Gln Gln Trp Ser Ser Asn Pro Arg Thr 1 5 <210> 101 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Dextran <400> 101 Asn Leu Val Pro Met Val Ala Thr Val 1 5

Claims

1. Use of a first modified T-cell and a second modified T-cell comprising a chimeric antigen receptor (CAR) in the preparation of a medicament for treating cancer in a subject in need thereof, wherein in the first modified T-cell the CAR comprises an antigen-binding domain capable of binding CD5, a transmembrane domain and an intracellular domain, and in the second modified T-cell the endogenous CD5 gene has been knocked out, wherein the cancer comprises T-cell lymphoma or T-cell leukemia.

2. The use according to claim 1, wherein the endogenous CD5 gene is knocked out using the CRISPR method.

3. The use according to claim 2, wherein the CRISPR method is the CRISPR / Cas9 method.

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

23.

5. The use according to claim 1, wherein the antigen-binding domain of the CAR comprises complementarity-determining regions (CDRs), the complementarity-determining regions (CDRs) comprising amino acid sequences selected from SEQ ID NO:84-88 and 95-100.

6. The use according to claim 1, wherein the antigen-binding domain of the CAR comprises a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:75, 81 and 93.

7. The use according to claim 6, wherein the antigen-binding domain of the CAR comprises a light chain variable region, the light chain variable region comprising an amino acid sequence selected from SEQ ID NO:76, 82 and 94.

8. The use according to claim 1, wherein the antigen-binding domain of the CAR comprises a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

75.

9. The use according to claim 1, wherein the antigen-binding domain of the CAR comprises a light chain variable region, the light chain variable region comprising the amino acid sequence of SEQ ID NO:

76.

10. The use according to claim 1, wherein the antigen-binding domain of the CAR comprises a scFv, the scFv comprising amino acid sequences selected from SEQ ID NO:73, 74, 79, 80, 91 and 92.

11. The use according to claim 1, wherein the antigen-binding domain of the CAR comprises a scFv, the scFv comprising the amino acid sequence of SEQ ID NO:

73.

12. The use according to claim 1, wherein the antigen-binding domain of the CAR comprises a scFv, the scFv comprising the amino acid sequence of SEQ ID NO:

74.

13. The use according to claim 1, wherein the CAR comprises amino acid sequences selected from SEQ ID NO:71, 72, 77, 78, 89 and 90.

14. The use according to claim 1, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

72.

15. The use according to any one of claims 1 to 14, wherein the T cell lymphoma or T cell leukemia is selected from acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

16. The use according to claim 15, wherein the acute lymphoblastic leukemia (ALL) is T cell acute lymphoblastic leukemia (T-ALL).

17. The use according to any one of claims 1 to 16, wherein the CAR further comprises a suicide gene.

18. The use according to claim 17, wherein the suicide gene is iCaspase9.

19. A composition comprising a first modified T-cell, the first modified T-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 T-cell, wherein the endogenous CD5 gene has been knocked out.

20. A composition comprising a first modified T-cell, the first modified T-cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain capable of binding CD5, a transmembrane domain, and an intracellular domain, and a second modified T-cell, wherein the endogenous CD5 gene has been knocked out, wherein the antigen-binding domain of the CAR is encoded by a nucleic acid sequence selected from SEQ ID NO: 8-13.

21. The composition according to claim 20, further comprising a pharmaceutically acceptable carrier.

22. The composition according to claim 20 or 21, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 71, 72, 77, 78, 89, and 90.

23. The composition according to claim 20 or 21, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

72.

24. A pharmaceutical composition comprising a population of T cells, wherein the population comprises a first population of cells containing a chimeric antigen receptor (CAR) and a second population of cells containing a mutated endogenous CD5 gene, wherein the CAR comprises an antigen-binding domain capable of binding CD5, wherein the antigen-binding domain comprises complementarity-determining regions (CDRs), the complementarity-determining regions (CDRs) comprising amino acid sequences selected from SEQ ID NO: 84-88 and 95-100, and wherein the mutated endogenous CD5 gene is a gene-edited endogenous CD5 gene; and wherein the population of cells containing the mutated endogenous CD5 gene has reduced expression of endogenous CD5 protein.

25. The pharmaceutical composition according to claim 24, wherein the population of cells comprises more than one chimeric antigen receptor.

26. The pharmaceutical composition according to claim 24, wherein the mutated CD5 gene is a CRISPR-mutated CD5 gene.

27. The pharmaceutical composition according to claim 24, wherein the antigen-binding domain of the CAR comprises a heavy-chain variable region, and the heavy-chain variable region comprises an amino acid sequence selected from SEQ ID NO: 75, 81, and 93.

28. The pharmaceutical composition according to claim 27, wherein the antigen-binding domain comprises a light-chain variable region, and the light-chain variable region comprises an amino acid sequence selected from SEQ ID NO: 76, 82, and 94.

29. The pharmaceutical composition according to claim 24, wherein the antigen-binding domain of the CAR comprises a heavy-chain variable region, and the heavy-chain variable region comprises the amino acid sequence of SEQ ID NO:

75.

30. The pharmaceutical composition according to claim 24, wherein the antigen-binding domain of the CAR comprises a light-chain variable region, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO:

76.

31. The pharmaceutical composition according to claim 24, wherein the antigen-binding domain of the CAR comprises a scFv, and the scFv comprises an amino acid sequence selected from SEQ ID NO: 73, 74, 79, 80, 91, and 92.

32. The pharmaceutical composition according to claim 24, wherein the antigen-binding domain of the CAR comprises a scFv, and the scFv comprises the amino acid sequence of SEQ ID NO:

73.

33. The pharmaceutical composition according to claim 24, wherein the antigen-binding domain of the CAR comprises a scFv, and the scFv comprises the amino acid sequence of SEQ ID NO:

74.

34. The pharmaceutical composition according to claim 24, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 71, 72, 77, 78, 89, and 90.

35. The pharmaceutical composition according to claim 24, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

72.

36. Use of the pharmaceutical composition according to claim 24 in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the cancer comprises T cell lymphoma or T cell leukemia.

37. The use according to claim 36, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

38. The use according to claim 37, wherein the acute lymphoblastic leukemia (ALL) is T cell acute lymphoblastic leukemia (T-ALL).

39. Use of a first modified T cell comprising a mutated endogenous CD5 gene and a second modified T cell comprising a chimeric antigen receptor in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the chimeric antigen receptor comprises an antigen-binding domain capable of binding CD5, wherein the use enhances the efficacy of the T cells in the subject, and wherein the mutated endogenous CD5 gene is a knockout of the endogenous CD5 gene.

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