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

Abstract

The present invention provides compositions and methods for treating T-cell lymphoma and T-cell leukemia. [Solution] A method for treating cancer comprising the steps of administering a first modified cell containing a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain to the subject, and administering a second modified cell in which the endogenous CD5 gene is knocked out to the subject. Preferably, the antigen-binding domain is an antigen-binding domain that can bind to CD2, CD5, or CD7, and preferably the endogenous CD5 gene is knocked out by the CRISPER / Cas9 method.

Description

[Technical Field]

[0001] Cross-reference of related applications This application has priority under 35 U.S.C. § 119(e) based on U.S. Provisional Patent Application No. 62 / 782,131, filed on 19 December 2018, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention T-cell lymphomas and T-cell leukemias are invasive neoplasms derived from T-cell progenitor cells or differentiated T cells. Mature T-cell lymphomas or peripheral T-cell lymphomas account for 10% to 15% of all non-Hodgkin lymphomas, or approximately 7,000 to 10,000 cases per year in the United States. T-cell lymphomas and T-cell leukemias have a poor prognosis and few treatment options are available. Chimeric antigen receptor T-cell (CART) therapy has shown efficacy against B-cell neoplasms, but extending the success of CAR T-cells to T-cell malignancies is difficult because most target antigens are shared between normal and malignant cells, leading to sibling killing of CAR T cells.

[0003] There is a need for compositions and methods for treating T-cell lymphoma and T-cell leukemia, as well as for methods to eliminate sibling killing of CAR T cells. This invention solves this need. [Overview of the Initiative]

[0004] As described herein, the present invention relates to compositions and methods utilizing CAR T cells that target CD2, CD5, or CD7, and modified cells in which CD2, CD5, or CD7 is knocked out.

[0005] In one aspect, the present invention includes a method for treating cancer in a subject requiring such treatment. The method includes administering a first modified cell to a subject that contains a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, and administering a second modified cell to a subject in which the endogenous CD5 gene is knocked out.

[0006] In another aspect, the present invention includes a method for treating cancer in a subject that requires it. The method includes administering to a subject a first modified cell comprising a chimeric antigen receptor (CAR) having an antigen-binding domain, a transmembrane domain and an intracellular domain that can bind to CD2, and administering to a subject a second modified cell in which the endogenous CD2 gene is knocked out.

[0007] In yet another aspect, the present invention includes a method for treating cancer in a subject that requires it. The method includes administering to a subject a first modified cell comprising a CAR having an antigen-binding domain, a transmembrane domain and an intracellular domain capable of binding to CD5, and administering to a subject a second modified cell in which the endogenous CD5 gene is knocked out.

[0008] In yet another aspect, the present invention includes a method for treating cancer in a subject requiring such treatment, comprising the steps of administering to a subject a first modified cell comprising a CAR having an antigen-binding domain, a transmembrane domain and an intracellular domain capable of binding to CD7, and administering to a subject a second modified cell in which the endogenous CD7 gene is knocked out.

[0009] Another aspect of the present invention relates to nucleic acids comprising a CAR having an antigen-binding domain, a transmembrane domain, and an intracellular domain that can bind to CD2.

[0010] Another aspect of the present invention relates to nucleic acids comprising a CAR having an antigen-binding domain, a transmembrane domain, and an intracellular domain that can bind to CD5.

[0011] Another aspect of the present invention is a vector comprising any of the nucleic acids disclosed herein.

[0012] In another aspect, the present invention includes a cell comprising any of the nucleic acids disclosed herein or any of the vectors disclosed herein.

[0013] In yet another aspect, the present invention includes a composition comprising a first modified cell containing a chimeric antigen receptor (CAR) comprising a CD2-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD2 gene is knocked out.

[0014] In yet another aspect, the present invention includes a composition comprising a first modified cell containing a chimeric antigen receptor (CAR) comprising a CD5-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD5 gene is knocked out.

[0015] In another aspect, the present invention includes a composition comprising a first modified cell containing a chimeric antigen receptor (CAR) comprising a CD7-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD7 gene is knocked out.

[0016] Another aspect of the present invention includes a composition comprising a first modified cell containing a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD5 gene is knocked out.

[0017] In various aspects of the above-mentioned aspects of the present invention or any other aspects, endogenous genes are knocked out using the CRISPR method. In one aspect, the CRISPR method is the CRISPR / Cas9 method. In one aspect, the CRISPR / Cas9 method utilizes an sgRNA containing a nucleotide sequence of SEQ ID NO:23. In one aspect, the CRISPR / Cas9 method utilizes an sgRNA containing a nucleotide sequence selected from the group consisting of SEQ ID NO:22-24.

[0018] In one embodiment, the antigen-binding domain of CAR is CD5, CD19, CD2, CD7, tumor-specific antigen (TSA), tumor-associated antigen (TAA), glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, 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, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, MART-1 / Melan A (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBV A, HPV antigen E6, HPV antigen E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p It can bind to antigens selected from the group consisting of 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA15-3, CA27.29, BCAA, CA195, CA242, 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, cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0019] In one embodiment, the antigen-binding domain of the CAR includes a complementation-determining region (CDR) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36, 43-48, 53-58, 65-70, 83-88, and 95-100. In one embodiment, the antigen-binding domain of the CAR includes a complementation-determining region (CDR) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36, 43-48, 53-58, and 65-70. In one embodiment, the antigen-binding domain of the CAR includes a complementation-determining region (CDR) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-88 and 95-100.

[0020] In one embodiment, the antigen-binding domain of the CAR includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 41, 51, 63, 75, 81, and 93. In another embodiment, the antigen-binding domain of the CAR includes a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 42, 52, 64, 76, 82, and 94.

[0021] In one embodiment, the antigen-binding domain of the CAR includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 41, 51, and 63. In another embodiment, the antigen-binding domain of the CAR includes a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 42, 52, and 64.

[0022] In one embodiment, the antigen-binding domain of the CAR includes a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 75, 81, and 93. In another embodiment, the antigen-binding domain of the CAR includes a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 82, and 94.

[0023] In certain embodiments, the antigen-binding domain of the CAR comprises a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79, 80, 91, and 92. In certain embodiments, the antigen-binding domain of the CAR comprises a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 28, 39, 40, 50, 61, and 62. In certain embodiments, the antigen-binding domain of the CAR comprises a scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 73, 74, 79, 80, 91, and 92.

[0024] In certain embodiments, the CAR comprises an amino acid sequence selected from the group consisting of 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 the group consisting of SEQ ID NO: 25, 26, 37, 38, 49, 59, and 60. In certain embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 71, 72, 77, 78, 89, and 90.

[0025] In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-13. In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-7. In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8-13.

[0026] In certain embodiments, the CAR further comprises a suicide gene. In certain embodiments, the suicide gene is iCaspase9.

[0027] In certain embodiments, the first and second modified cells are T cells.

[0028] In one embodiment, cancer includes T-cell lymphoma or T-cell leukemia. In one embodiment, cancer is selected from the group consisting of acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0029] In one embodiment, the composition comprises a pharmaceutically acceptable carrier. [Brief explanation of the drawing]

[0030] The following detailed description of specific embodiments of the present invention will be better understood when viewed in conjunction with the accompanying drawings. For illustrative purposes, the drawings show exemplary embodiments. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings.

[0031] [Figure 1] This is a schematic diagram illustrating the current problems with CART therapy for T-cell neoplasms. [Figure 2] This schematic diagram illustrates the development of an innovative strategy in which (i) tumor targets are removed from normal T cells using gene editing, thereby avoiding fraternal killing during production; and (ii) a second T cell product containing normal T cells with knocked-out (KO) T cell targets is co-injected with anti-T cell neoplasm CART to provide T cell immunity unaffected by CART death. [Figure 3] This schematic diagram illustrates a novel approach to targeting T-cell lymphoma without causing T-cell damage. A dual immunotherapy approach is employed, involving anti-T-NHL (or T-ALL) CART (either CART2 / 5 / 7, e.g., CART2) and CD2 / 5 / 7 knockout normal T cells. CART destroys tumor cells but also kills normal T cells. Infusion of CD2 / 5 / 7 (or other tumor-targeted) knockout normal T cells provides CART-resistant T-cell immunity until the CART cells are depleted. [Figure 4]The anti-CD2 CAR constructs and anti-CD5 CAR constructs used herein are shown. All constructs have a lentivirus pTRPE 4-1BB CD3ζ skeleton. [Figure 5] This study demonstrates the efficiency of CART transduction in T cells. Six different CAR5 constructs were generated using single-chain variable fragments (scFv) with high affinity (#17), moderate affinity (#34), and low affinity (#9). T cells were activated with anti-CD3 / CD28 beads (Dynabeads), and after 24 hours, 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 less than 350 fl. CAR expression (goat anti-mouse Fab antibody) was tested on day 6. [Figure 6] This shows the CD5 (or CD2 or CD7) KO manufacturing process and CRISPR-Cas9 KO efficiency. [Figure 7] The expansion curves for several CART groups are shown. Without CD2 knockout, CART2 cells did not expand. With knockout, CART2 and CART5 cells doubled by approximately 5–8 populations. [Figure 8] Without CRISPR-Cas9 knockout of CD5, the mean fluorescence intensity (MFI) of CD5 was 10 times lower in CART5 compared to control T cells, but there were no changes in other pan-T cell markers such as CD2. [Figure 9] The enlarged curves for CART2 and CART5 are shown. T cell concentrations were measured using a Coulter Counter. [Figure 10] The results from experiments in which six different CAR2 constructs were co-cultured with luciferase-+Jurkat cells (T-cell leukemia cell line) in vitro are shown. Total cell death was measured at 24 hours as a relative decrease in luminescence. Only C3029, C3030, and C3043 showed antitumor effects. [Figure 11]The results from in vitro challenges involving co-culture of six different CAR5 constructs with luciferase-+Jurkat cells (T-cell leukemia cell line) are shown. Total cell death was measured at 24 hours as a relative decrease in luminescence. All CAR5 constructs showed similar antitumor effects. [Figure 12] This study demonstrates the in vivo efficacy of CART2 and CART5. NSG mice were transplanted with luciferase-+Jurkat cells, and on day 7, mice were randomized to receive either control T cells or CART2 or CART5 (1 × 10⁶). Mice were imaged weekly using IVIS Xenogen Spectrum and analyzed with LivingImage software. CART2 C3043 and CART5 C3054 were the most effective. [Figure 13] Results from experiments in which Jurkat cells were transduced with different CAR5 constructs (targeted epitopes and affinities shown on the left) and a GFP-NFAT reporter, and then co-cultured with CD5+ tumor cells (or controls) for 24 hours are shown. Lead CART5 (C3054) showed increased NFAT activation. [Figure 14] The results from experiments in which Jurkat cells were transduced with different CAR2 constructs and GFP-NFAT reporters, and then co-cultured with CD2+ tumor cells (or controls) for 24 hours are shown. Lead CART2 (C3043) showed increased NFAT activation. [Figure 15] This study demonstrates the activity of CART2 and CART5 against cutaneous T-cell lymphoma. The results of a 24-hour cell death assay are shown. CART2 cells were active against primary Sézary cells (leukemic cutaneous T-cell lymphoma) and HH Sézary cell lines. CART5 was also active against HH cells. [Figure 16] The findings suggest that CART2 and CART5 can recognize and destroy normal T cells (self (top) and allogeneic (bottom)). [Figure 17]The findings suggest that removal of CAR targets protects normal T cells from CART-mediated death. CD5 knockout cells are resistant to CART5-mediated death, while WT normal T cells are not. Normal resting T cells are recognized and killed by CART2 (top) and CART5 (bottom). Efficient knockout of CD2 or CD5 from normal T cells using CRISPR-Cas9 results in resistance to CART2 or CART5-mediated death, respectively. [Figure 18] The study demonstrates the presence of CMV-specific T cells in the normal T cell products of CD2KO and CD5KO mutants. Normal T cells from CD2KO and CD5KO mutants maintain the ability to recognize CMV peptides and produce cytokines. (HLA-A-02:01-CMV PP65 NLVPMVATV dextramer (SEQ ID NO: 101); ICS after 4h exposure to CETF peptide, followed by secondary culture with CMV-peptide pulsed APC). [Figure 19] This report demonstrates the development of bispecific CAR T cells. Two lentiviral constructs containing CAR5 (C3054) and CAR2 (C3043), linked by a P2A sequence, were generated. Gene expression is driven by the EF1α promoter. The CAR5 construct possesses a 4-1BB costimulatory domain and a CD3ζ signaling domain. [Figure 20A] Figures 20A–20B show dual KO CART cells, demonstrating efficient knockout of both CD2 and CD5 in normal T cells, as shown by flow cytometry. [Figure 20B] Please refer to the explanation in Figure 20A. [Figure 21] The findings suggest that CD5 KO CART5 is more effective than CD5+ CART5 in vivo. CD5 KO enhances the antitumor efficacy of CART5. In a Jurkat T-ALL xenograft model using NSG mice, CD5 KO CART5 (2 × 10⁶ cells / mouse) resulted in a complete long-term response and longer survival compared to WT CART5. [Figure 22]CD5 knockout CART19 is shown to be more effective than CD5 + CART19 in vivo. CD5 knockout enhances the antitumor efficacy of CART19. In a NALM6 B-ALL xenograft model, CD5 knockout CART19 shows significantly better tumor control compared to WT CART19. [Figure 23] Figures 23A and 23B show findings that CART5 and CART2 can target 20% of AML. Figure 23A shows CD2 expression in AML. Figure 23B shows results from a 24-hour cell death assay. CART2 cells were co-cultured with CD2+ AML cells and showed significant cell death at 24 hours. [Figure 24] The findings suggest that CART5 can target 100% of CLL and MCL. The results come from cytotoxicity assays showing that CART5 cells can recognize and kill CD5+ MCL cell lines (Jeko-1 and Mino). [Figure 25] This paper (above) demonstrates the development of sgRNA for knocking out CD7 in T cells and the generation of six CAR constructs targeting CD7. [Figure 26] The Casp9-CAR5 lentiviral constructs used herein are shown. Two lentiviral constructs were generated, each containing either 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 costimulatory domain and a CD3ζ signaling domain. [Modes for carrying out the invention]

[0032] Detailed explanation definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in the practice of testing of the present invention, but preferred materials and methods are described herein. The following technical terms are used to describe and assert the present invention:

[0033] It should be understood that the technical terms used in this specification are intended to describe only specific aspects and are not intended to limit them.

[0034] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one or more elements.

[0035] As used herein with respect to measurable values ​​such as quantity and duration, the term "about" is intended to include a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, because such variation is appropriate for carrying out the disclosed method.

[0036] As used herein, “activation” refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation may also relate to induced cytokine production and detectable effector function. The term “activated T cell” refers, in particular, to a T cell undergoing cell division.

[0037] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be intact immunoglobulins derived from natural or recombinant sources, or they may be the immunoreactive portion of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in this invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single-chain antibodies (scFv) 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).

[0038] The term "antibody fragment" refers to a portion of an intact antibody, specifically the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0039] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0040] As used herein, "antibody light chain" refers to the smaller of two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. κ and λ light chains refer to the two main antibody light chain isotypes.

[0041] As used herein, the term "synthetic antibody" means an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage as described herein. This term should also be considered to mean an antibody produced by the synthesis of an antibody protein or an antibody-coding DNA molecule expressing the amino acid sequence that defines that antibody, wherein the DNA sequence or amino acid sequence is obtained using a synthesis technique for DNA sequences or amino acid sequences that are available and well known in the art.

[0042] As used herein, the terms “antigen” or “Ag” are defined as molecules that induce an immune response. This immune response may include either or both antibody production or activation of specific immune-qualified cells. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can act as an antigen. Furthermore, antigens may be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, therefore, encodes an “antigen” as the term is used herein. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention involves, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences can be arranged in various combinations to induce a desired immune response. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by a “gene.” It is readily apparent that antigens may be made from, synthesized from, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0043] As used herein, the term “self” is intended to mean any material derived from the same individual that is later reintroduced into that individual.

[0044] "Same species" refers to any substance that originates from different animals of the same species.

[0045] "Different species" refers to any substance derived from an animal of a different species.

[0046] The terms “chimeric antigen receptor” or “CAR” as used herein refer to an artificial T cell receptor that is expressed in immune effector cells and modified to specifically bind to an antigen. CARs can be used therapeutically by adoptive cell transfer. T cells are removed from a patient and modified to express a receptor specific to a particular type of antigen. In some embodiments, CARs have specificity to a selected target, e.g., B cell surface receptors. CARs may also include an extracellular domain containing an intracellular activation domain, a transmembrane domain, and a tumor-associated antigen-binding region. In some aspects, CARs include an extracellular domain containing an anti-B cell-binding domain fused with a CD3ζ transmembrane domain and an intracellular domain.

[0047] The term "cleavage" refers to the breakdown of covalent bonds in the backbone of nucleic acid molecules, or the hydrolysis of peptide bonds. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand breaks are possible. Double-strand breaks can occur as a result of two different single-strand break events. DNA breaks can result in the formation of either blunt or adherent ends. In some embodiments, fusion polypeptides can be used to target cleaved double-stranded DNA.

[0048] As used herein, the term “conservative sequence modification” is intended to mean an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of an antibody can be replaced with other amino acid residues from the same side chain family, and this modified antibody can be tested for antigen-binding ability using the functional assay methods described herein.

[0049] When this term is used herein, “co-stimulatory ligand” includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to a congeneral co-stimulatory molecule on a T cell, thereby providing signals that mediate T cell responses, including but not limited to proliferation, activation, and differentiation, in addition to the primary signal provided by, for example, the binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Co-stimulatory ligands may 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), intracellular 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 to Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3, as well as ligands that specifically bind to CD83.

[0050] A "costimulatory molecule" is a congeneral binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by the T cell, such as proliferation, but is not limited to these. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0051] As used herein, “co-stimulatory signal” refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0052] A "disease" is a health condition in which an animal is unable to maintain homeostasis, and its health will continue to deteriorate unless the disease is treated. In contrast, a "disorder" in an animal is a health condition in which the animal can maintain homeostasis, but its health is less desirable than when it is not disordered. Leaving a disorder untreated does not necessarily lead to a further decline in the animal's health.

[0053] As used herein, the term "downregulation" refers to a reduction or loss of expression of one or more genes.

[0054] "Effective dose" or "therapeutic effective dose" is used interchangeably herein and refers to the amount of a compound, formulation, material, or composition described herein that is effective in achieving a particular biological outcome or that provides a therapeutic or preventive benefit. Such outcomes may include, but are not limited to, antitumor activity as determined by any appropriate means in the art.

[0055] "Code" refers to the inherent properties and resulting biological characteristics of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence, and which acts as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, a gene codes for a protein when the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and is usually listed in sequence listings, and the non-coding strand, which is used as a template for the transcription of a gene or cDNA, can be said to code for a protein, or other products of that gene or cDNA.

[0056] As used herein, “endogenous” means any material that originates from or is produced within an organism, cell, tissue, or system.

[0057] As used herein, the term “exogenous” means any material introduced from or produced outside of an organism, cell, tissue, or system.

[0058] As used herein, the term “expand” means an increase in number, such as an increase in the number of T cells. In one embodiment, T cells expanded ex vivo increase in number compared to the number initially present in the culture. In another embodiment, T cells expanded ex vivo increase in number compared to other cell types in the culture. As used herein, the term “ex vivo” means cells taken from an organism (e.g., human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0059] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0060] An "expression vector" is a vector containing recombinant polynucleotides that include an expression regulatory sequence functionally linked to the nucleotide sequence to be expressed. An 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 known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus and adeno-associated virus) incorporating recombinant polynucleotides.

[0061] As used herein, “homology” means the subunit sequence identity between two polymer molecules, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. Homologous to two molecules is when the subunit positions in both molecules are occupied by the same monomeric subunit; for example, if the positions in each of two DNA molecules are occupied by adenine, then they are homologous at those positions. Homologousness between two sequences is a linear function of the number of identical or homologous positions; for example, if half of the positions in two sequences (e.g., five positions in a polymer with a length of 10 subunits) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are identical or homologous, then the two sequences are 90% homologous.

[0062] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in either the recipient antibody or the introduced CDR or framework sequence. These modifications are made to further improve and optimize antibody performance. Generally, humanized antibodies contain at least one, and typically two, substantially all, of the variable domains, where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to that of a human immunoglobulin sequence. Humanized antibodies also optimally contain at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0063] "Completely human" refers to immunoglobulins, such as antibodies, whose entire molecule is of human origin, or whose amino acid sequence is identical to that of human antibodies.

[0064] As used herein, “identity” refers to the subunit sequence identity between two polymer molecules, such as between two polypeptide molecules, and especially between two amino acid molecules. Two amino acid sequences are identical if they have the same residue at the same position; for example, if a position in each of two polypeptide molecules is occupied by arginine, then they are identical at that position. The degree to which two amino acid sequences have the same residue at the same position in alignment, or their identity, is often expressed as a percentage. The identity between two amino acid sequences is a linear 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 10 amino acids long) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matching or identical, then the two amino acid sequences are 90% identical.

[0065] As used herein, the terms “immunoglobulin” or “Ig” are defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody found in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most common antibody in circulating blood. IgM is the major immunoglobulin produced in the primary immune response in most targets. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin whose antibody function is unknown, but it may act as an antigen receptor. IgE is an immunoglobulin that mediates immediate-type hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0066] As used herein, the term “immune response” is defined as a cellular response to an antigen that occurs when lymphocytes recognize an antigen molecule as a foreign substance, induce antibody formation, and / or activate lymphocytes to eliminate the antigen.

[0067] Where an "immunologically effective dose" or "therapeutic dose" is indicated, the exact amount of the composition of the present invention to be administered may be determined by a physician or researcher, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition.

[0068] Where used herein, “instructional material” includes publications, records, diagrams, or any other expressive medium that can be used to convey the usefulness of the compositions and methods of the present invention. The instructional material for the kit of the present invention may, for example, be attached to the container containing the nucleic acids, peptides, and / or compositions of the present invention, or may be shipped together with the container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and compounds be used jointly by the recipient.

[0069] "Isolated" means that something has been altered or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from its natural coexisting material is "isolated." Isolated nucleic acids or proteins can exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0070] As used herein, the term "knockdown" refers to a reduction in the gene expression of one or more genes.

[0071] As used herein, the term "knockout" refers to the loss of gene expression in one or more genes.

[0072] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they are one of the most efficient methods of gene delivery vectors because they can deliver a significant amount of genetic information into the host cell's DNA. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0073] As used herein, the term “limited toxicity” refers to peptides, polynucleotides, cells, and / or antibodies of the present invention that have been shown to have substantially negative biological effects, antitumor effects, and substantially negative physiological effects on healthy cells, non-tumor cells, non-disease cells, non-target cells, or populations of such cells, either in vivo or in vitro.

[0074] As used herein, the term "modified" means an altered state or structure of the molecules or cells of the present invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0075] As used herein, the term “modulate” means mediating a detectable increase or decrease in the level of response in a subject compared to the level of response in the subject in the absence of the treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject. This term encompasses, in a subject, preferably a human, disrupting and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response.

[0076] In the context of this invention, the following abbreviations for commonly existing nucleic acid bases are used: "A" refers to adenosine, "C" to cytosine, "G" to guanosine, "T" to thymidine, and "U" to uridine.

[0077] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding RNA or protein" also includes introns to the extent that protein-coding nucleotide sequences may contain introns, depending on the type.

[0078] The term "functionally linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, if a first nucleic acid sequence is positioned under a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is functionally linked to the coding sequence. In general, functionally linked DNA sequences are contiguous and, if it is necessary to link two protein-coding regions, they are in the same reading frame.

[0079] The terms “overexpressed” tumor antigen or “overexpression” of tumor antigen are intended to indicate that the expression of tumor antigen in cells from a diseased area, such as a solid tumor, within a particular tissue or organ of a patient is at an abnormal level compared to the level of expression in normal cells from that tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of tumor antigen can be determined by standard assay methods known in the art.

[0080] Parenteral administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion.

[0081] As used herein, the term “polynucleotide” is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have general knowledge that nucleic acids are polynucleotides and that they can be hydrolyzed to monomeric “nucleotides.” Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR®, and by synthetic means.

[0082] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, this term refers to both short chains, commonly known in the art as peptides, oligopeptides, and oligomers, and long chains, commonly known in the art as proteins, of which there are many types. Polypeptides include, among other things, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0083] As used herein, the term “promoter” is defined as a DNA sequence recognized by a cellular synthetic mechanism or introduced synthetic mechanism, which is necessary to initiate the specific transcription of a polynucleotide sequence.

[0084] A "signaling pathway" refers to the biochemical relationships between various signaling molecules that play a role in the transmission of signals from one part of a cell to another. The term "cell surface receptor" includes molecules and molecular complexes that can receive signals and transmit them across the cell's plasma membrane.

[0085] As used herein with respect to antibodies, the term “specifically binding” means an antibody that recognizes a specific antigen but substantially does not recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different alleles of that antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the terms “specific binding” or “specifically binding” may be used in relation to the interaction between an antibody, protein, or peptide and a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope); for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific to epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction involving labeled “A” and that antibody.

[0086] The term "stimulation" refers to a primary response induced by a stimulating molecule (e.g., the TCR / CD3 complex) binding to its homologous ligand, thereby mediating a signaling event, such as, but not limited to, signaling mediated by the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as the downregulation of TGF-β and / or rearrangement of the cytoskeleton.

[0087] When used herein, "stimulating molecule" means a molecule on a T cell that specifically binds to a homologous stimulating ligand present on an antigen-presenting cell.

[0088] As used herein, "stimulating ligand" means a ligand that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), specifically binds to a congenital binding partner on T cells (referred to herein as a "stimulating molecule"), thereby mediating a primary response by T cells, including but not limited to activation, initiation of an immune response, and proliferation. Stimulating ligands are well known in the art and include, in particular, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0089] The term “subject” is intended to include organisms (e.g., mammals) from which an immune response may be induced. As used herein, “subject” or “patient” may be human or a non-human mammal. Non-human mammals include livestock and pets, such as sheep, cattle, pigs, dogs, cats, and rodents. Preferably, the subject is human.

[0090] As used herein, “substantially purified” cells are cells that essentially contain no other cell types. Furthermore, substantially purified cells refer to cells isolated from other cell types that are normally associated with them in their native state. In some instances, a population of substantially purified cells refers to a homogeneous population of cells. In other instances, the term simply refers to cells isolated from cells that are normally associated with them in their native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0091] 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 for binding to occur.

[0092] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins involved in the activation of T cells in response to antigen presentation. The TCR is responsible for recognizing antigens bound to the major histocompatibility complex (MHI) molecule. The TCR is composed of a heterodimer of alpha (a) and beta (β) chains, although 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, namely a variable domain and a constant domain. In some embodiments, the TCR can be modified on any cell containing a TCR (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells).

[0093] As used herein, the term “therapeutic” means treatment and / or prevention. Therapeutic effects are obtained by suppression, remission, or eradication of the disease state.

[0094] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which exogenous nucleic acids are transferred to or introduced into host cells. “Transfected,” “transformed,” or “transduced” cells are those that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary target cells and their offspring.

[0095] When this term is used herein, “to treat” a disease means to reduce the frequency or severity of at least one sign or symptom of the disease or disorder that the subject is suffering from.

[0096] As used herein, the terms “transcriptionally controlled” or “functionally linked” mean that the promoter is in the correct position and orientation with respect to the polynucleotide in order to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0097] A “vector” is a composition containing isolated nucleic acids that can be used to deliver isolated nucleic acids into the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmidal and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors.

[0098] Scope: Throughout this disclosure, various aspects of the invention can be presented in the form of scope. It should be understood that the scope of scope is merely for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, scope descriptions should be considered to specifically disclose all possible sub-scopes and the individual numbers within those scopes. For example, a scope description such as 1-6 should be considered to specifically disclose sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numbers within those scopes, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0099] explanation This disclosure describes three chimeric antigen receptors (CARs) that target T-cell neoplasms and a method to prevent sibling killing of healthy T cells. T-cell lymphomas and T-cell leukemias are invasive neoplasms derived from T-cell progenitor cells or differentiated T cells. Mature T-cell lymphomas or peripheral T-cell lymphomas account for 10% to 15% of all non-Hodgkin lymphomas, or approximately 7,000 to 10,000 cases per year in the United States. T-cell lymphomas and T-cell leukemias have a poor prognosis and few treatment options are available. While CART therapy has shown efficacy against B-cell neoplasms, extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies is currently challenging because most target antigens are shared between normal and malignant cells, leading to sibling killing of CAR T cells. In this invention, CRISPR-Cas editing is used to remove target antigens from healthy T cells, protect them from CART cell therapy, and eliminate potentially fatal immunosuppression caused by the elimination of T cell compartments.

[0100] In one embodiment, CAR targets T cell antigens CD2, CD5, and CD7. In another embodiment, CRISPR-Cas knockout of the targeted CD2, CD5, or CD7 in healthy T cells prevents the death of healthy T cells during production and subsequent CART treatment.

[0101] Treatment method The present invention includes a method for treating T-cell lymphoma or T-cell leukemia in subjects where it is needed. In another aspect, the present invention includes a method for preventing fraternal killing of CAR T cells in subjects where it is needed.

[0102] In one embodiment, the method includes the steps of administering to a target a first modified cell containing a chimeric antigen receptor (CAR) comprising a CD2-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and administering to a target a second modified cell in which the endogenous CD2 gene is knocked out.

[0103] In one embodiment, the method includes the steps of administering to a target a first modified cell containing a CAR including a CD5-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and administering to a target a second modified cell in which the endogenous CD5 gene is knocked out.

[0104] In one embodiment, the method includes the steps of administering to a target a first modified cell containing a CAR including a CD7-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and administering to a target a second modified cell in which the endogenous CD7 gene is knocked out.

[0105] In one embodiment, the method includes the steps of administering a first modified cell containing a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain to a target, and administering a second modified cell in which the endogenous CD5 gene is knocked out to a target.

[0106] In one embodiment, the method includes the step of administering modified cells containing a chimeric antigen receptor (CAR) to a target, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and the endogenous CD5 gene is knocked out in the cell.

[0107] In various embodiments of the methods disclosed herein, the subject may be administered any of the CARs disclosed herein. The CARs may be specific to any tumor-associated antigen (TAA) or tumor-specific antigen (TSA) known to those skilled in the art.

[0108] In one embodiment, the CAR includes a complementarity-determining region (CDR) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36, 43-48, 53-58, 65-70, 83-88, and 95-100. In one embodiment, the CAR includes an antigen-binding domain containing a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 41, 51, 63, 75, 81, and 93, and / or a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 42, 52, 64, 76, 82, and 94. In one embodiment, the CAR includes an scFv containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79, 80, 91, and 92.

[0109] In one embodiment, the subject is administered a CAR containing a nucleic acid sequence encoded by one of SEQ ID NO: 1 to 13. In another embodiment, the CAR contains an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 37, 38, 49, 59, 60, 71, 72, 77, 78, 89, and 90.

[0110] In one embodiment, the first and / or second modified cells are T cells. In one embodiment, cancer includes T-cell lymphoma or T-cell leukemia. Types of cancer that can be treated by the compositions and methods of the present invention include, but are not limited to, non-Hodgkin lymphomas and their subtypes, such as peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL, ALK-), natural killer / T-cell lymphoma (NKTCL), adult T-cell leukemia / lymphoma (ATLL), ALCL ALK+, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like lymphoma, and unclassified PTCL.

[0111] In one embodiment, endogenous genes (e.g., CD2, CD5, and CD7) are knocked out using the CRISPR / Cas9 method. In one embodiment, the CRISPR / Cas9 method utilizes sgRNAs that target CD2, CD5, and / or CD7. In one embodiment, the sgRNAs contain nucleotide sequences selected from the group consisting of SEQ ID NO: 22-24.

[0112] In one embodiment, the CAR of the present invention further comprises a suicide gene. One non-limiting example of a suicide gene is the inducible caspase 9 gene (iCaspase9, iCasp9, or iC9). The iCaspase9 suicide gene system is based on the fusion of human caspase 9 with a modified human FK-binding protein, enabling conditional dimerization using a small molecule drug (e.g., AP1903). When exposed to a synthetic dimerizing agent, iCaspase9 is activated, leading to rapid apoptosis of cells expressing this 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 when expressed, it converts the non-toxic prodrug GCV into GCV triphosphate, leading to cell death by halting DNA replication.

[0113] composition One aspect of the present invention includes a composition comprising a first modified cell containing a chimeric antigen receptor (CAR) comprising a CD2-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD2 gene is knocked out.

[0114] Another aspect of the present invention includes a composition comprising a first modified cell containing a CAR having an antigen-binding domain targeting CD5, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD5 gene is knocked out.

[0115] A further aspect of the present invention includes a composition comprising a first modified cell containing a CAR comprising a CD7-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD7 gene is knocked out.

[0116] In one embodiment, the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 13. In one embodiment, the endogenous gene is knocked out using the CRISPR / Cas system. In one embodiment, the CRISPR / Cas9 system contains a gRNA containing a nucleic acid sequence selected from the group consisting of SEQ ID NO: 22 to 24.

[0117] The present invention further comprises the composition of the present invention and a pharmaceutically acceptable carrier.

[0118] Chimeric antigen receptor (CAR) The present invention provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain. In one embodiment, the present invention comprises a CAR comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain that can bind to CD2.

[0119] antigen-binding domain In one embodiment, the CAR of the present invention includes a target-specific binding element, also called 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, the antigen-binding domain may be selected to recognize ligands that act as cell surface markers on target cells associated with a specific disease condition (e.g., T-cell lymphoma or T-cell leukemia).

[0120] In one embodiment, the CAR of the present invention may be modified to target tumor antigens. The antigens described herein are included only as examples. The list is not exclusive, and further examples will be readily apparent to those skilled in the art. Tumor antigens are proteins produced by tumor cells that trigger an immune response, specifically a T cell-mediated immune response. The selection of antigen-binding domains of the present invention depends 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, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostain, PSMA, Her2 / neu, survivorbin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0121] The types of tumor antigens referred to in this invention may be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and are not present in other cells in the body. TAA-associated antigens are not unique to tumor cells and are expressed in normal cells under conditions that do not induce a state of immune tolerance to the antigen. Antigen expression in tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed in normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens normally present in normal cells at very low levels but expressed at much higher levels in tumor cells.

[0122] Non-limiting examples of TSA antigens or TAA antigens include: differentiation antigens, e.g., MART-1 / Melan A (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multisystem antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed fetal antigens, e.g., CEA; overexpressed oncogenes and mutant tumor suppressor genes, e.g., p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, e.g., 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, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, βHCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA This includes 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\cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0123] Depending on the desired antigen to be targeted, the CAR of the present invention may be modified to include an appropriate antigen-binding domain specific to the desired antigen target. For example, if CD2 is the desired antigen to be targeted, an antibody against CD2 can be used as the antigen-binding domain for incorporation into the CAR of the present invention.

[0124] In one embodiment, the antigen-binding domain of the CAR targets CD2. In another embodiment, the antigen-binding domain of the CAR targets CD5. In another embodiment, the antigen-binding domain of the CAR targets CD7.

[0125] In some embodiments, the antigen-binding domain within the CAR of the present invention is anti-CD2 scFV. In some embodiments, the antigen-binding domain within the CAR of the present invention is anti-CD5 scFV. In some embodiments, the antigen-binding domain within the CAR of the present invention is 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.

[0126] In one embodiment, the antigen-binding domain includes a heavy chain variable region containing three heavy chain complementarity-determining regions (HCDRs) and a light chain variable region containing three light chain complementarity-determining regions (LCDRs).

[0127] In one embodiment, the present invention comprises a CAR comprising an antigen-binding domain capable of binding to CD2, wherein the antigen-binding domain comprises a complementation-determining region (CDR) comprising one amino acid sequence 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.

[0128] In one embodiment, the CAR comprises an antigen-binding domain capable of binding to CD2, where 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.

[0129] In one embodiment, the CAR comprises an antigen-binding domain capable of binding to CD2, where 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.

[0130] In one embodiment, the CAR comprises an antigen-binding domain capable of binding to CD2, where 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.

[0131] In one embodiment, the CAR includes an antigen-binding domain capable of binding to CD2, wherein the antigen-binding domain includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 30. In one embodiment, the antigen-binding domain includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 42. In one embodiment, the antigen-binding domain includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 51 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 52. In one embodiment, the antigen-binding domain includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 63 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 64.

[0132] In one embodiment, the CAR comprises an antigen-binding domain capable of binding to CD2, wherein the antigen-binding domain is an scFv containing the amino acid sequence described in any one of SEQ ID NO: 27, 28, 39, 40, 50, 61, or 62.

[0133] In one embodiment, the present invention comprises a CAR comprising an antigen-binding domain capable of binding to CD5, wherein the antigen-binding domain comprises a complementation-determining region (CDR) comprising one amino acid sequence of SEQ ID NO: 83, 84, 85, 86, 87, 88, 95, 96, 97, 98, 99, or 100.

[0134] In one embodiment, the CAR comprises an antigen-binding domain capable of binding to CD5, where HCDR1 comprises the amino acid sequence of SEQ ID NO: 83, HCDR2 comprises the amino acid sequence of SEQ ID NO: 84, HCDR3 comprises the amino acid sequence of SEQ ID NO: 85, LCDR1 comprises the amino acid sequence of SEQ ID NO: 86, LCDR2 comprises the amino acid sequence of SEQ ID NO: 87, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 88.

[0135] In one embodiment, the CAR comprises an antigen-binding domain capable of binding to CD5, where HCDR1 comprises the amino acid sequence of SEQ ID NO:95, HCDR2 comprises the amino acid sequence of SEQ ID NO:96, HCDR3 comprises the amino acid sequence of SEQ ID NO:97, LCDR1 comprises the amino acid sequence of SEQ ID NO:98, LCDR2 comprises the amino acid sequence of SEQ ID NO:99, and LCDR3 comprises the amino acid sequence of SEQ ID NO:100.

[0136] In one embodiment, the CAR comprises an antigen-binding domain capable of binding to CD5, wherein the antigen-binding domain comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:75 and / or a light chain variable region containing the amino acid sequence of SEQ ID NO:76. In one embodiment, the heavy chain variable region contains the amino acid sequence of SEQ ID NO:81 and / or the light chain variable region contains the amino acid sequence of SEQ ID NO:82. In one embodiment, the heavy chain variable region contains the amino acid sequence of SEQ ID NO:93 and / or the light chain variable region contains the amino acid sequence of SEQ ID NO:94.

[0137] In one embodiment, the CAR comprises an antigen-binding domain capable of binding to CD5, and the antigen-binding domain is an scFv containing the amino acid sequence described in any one of SEQ ID NO: 73, 74, 79, 80, 91, or 92.

[0138] Acceptable 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 is at least one of the amino acid sequences described 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. It includes amino acid sequences having sequence identity of 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%.

[0139] transmembrane domain With respect to the transmembrane domain, the CAR may be designed to include a transmembrane domain fused with the extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains within the CAR is used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to minimize interaction with other members of the receptor complex, or to avoid such domain binding to the transmembrane domains of the same or different surface membrane proteins.

[0140] The transmembrane domain may be of natural origin or of synthetic origin. If the origin is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions particularly useful in the present invention may be derived from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154 (i.e., including at least their transmembrane regions). Alternatively, the transmembrane domain may be synthetic, in which case it mainly comprises hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, preferably, a short oligopeptide or polypeptide linker, 2 to 10 amino acids in length, may form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine doublets provide particularly suitable linkers.

[0141] In one embodiment, the transmembrane domain within the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain contains a nucleic acid sequence with SEQ ID NO: 14. In one embodiment, the CD8 transmembrane domain contains a nucleic acid sequence encoding an amino acid sequence with SEQ ID NO: 15. In another embodiment, the CD8 transmembrane domain contains an amino acid sequence with SEQ ID NO: 15.

[0142] In some embodiments, the transmembrane domain of the CAR of the present invention includes a CD8α hinge region. In one embodiment, the CD8 hinge domain includes a nucleic acid sequence with SEQ ID NO:16. In another embodiment, the CD8 hinge domain includes a nucleic acid sequence encoding an amino acid sequence with SEQ ID NO:17. In yet another embodiment, the CD8 hinge domain includes an amino acid sequence with SEQ ID NO:17.

[0143] A spacer domain may be incorporated between the antigen-binding domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term “spacer domain” generally means any oligopeptide or polypeptide that functions to link the transmembrane domain to either the extracellular domain or the cytoplasmic domain within the polypeptide chain. The spacer domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, most preferably 25 to 50 amino acids.

[0144] intracellular domain The intracellular or cytoplasmic domain of the CAR of the present invention is responsible for activating at least one of the normal effector functions of the immune cell to which the CAR is located. The term "effector function" refers to a specific function of the cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity including cytokine secretion. Therefore, the term "intracellular domain" means the portion of the protein that transmits the effector function signal and instructs the cell to perform the specific function. Usually, the entire intracellular domain may be used, but in many cases, it is not necessary to use the entire chain. Insofar as a shortened portion of the intracellular domain is used, such shortened portion may be used in place of the complete chain as long as it transmits the effector function signal. Therefore, the term intracellular domain shall include a shortened portion of the intracellular domain that is sufficient to transmit the effector function signal.

[0145] Preferred examples of intracellular domains for use in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act in coordination to initiate signal transduction after antigen receptor engagement, as well as derivatives or variants and synthetic sequences of these sequences having identical functional capabilities.

[0146] It is known that the signals generated through the TCR alone are insufficient for the complete activation of T cells, and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0147] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex either stimulatively or inhibitorily. Stimulative primary cytoplasmic signaling sequences may contain signaling motifs known as immunoreceptor-activating tyrosine motifs or ITAMs.

[0148] Examples of ITAMs containing primary cytoplasmic signaling sequences particularly useful in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. The cytoplasmic signaling molecule within the CAR of the present invention is particularly preferably a cytoplasmic signaling sequence derived from CD3ζ.

[0149] In a preferred embodiment, the intracellular domain of the CAR may be designed to include a CD3ζ signaling domain, either alone or in combination with other desired intracellular domains useful for the CAR of the present invention. For example, the intracellular domain of the CAR may include a portion of the CD3ζ chain and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen. Examples of such molecules include, but are not limited to, ligands that specifically bind to 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 CD83. Accordingly, while the present invention is primarily illustrated using 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of the present invention.

[0150] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention may be linked to each other in a random or specified order. Optionally, preferably, short oligopeptides or polypeptides, 2 to 10 amino acids in length, may form linkers. Glycine-serine doublets provide particularly suitable linkers.

[0151] In one embodiment, the intracellular domain is designed to include a CD3ζ signaling domain and a CD28 signaling domain. In another embodiment, the intracellular domain is designed to include a CD3ζ signaling domain and a 4-1BB signaling domain. In yet another embodiment, the intracellular domain is designed to include a CD3ζ signaling domain as well as CD28 and 4-1BB signaling domains.

[0152] In one embodiment, the intracellular domain within the CAR of the present invention is designed to include a 4-1BB signaling domain and a CD3ζ signaling domain, wherein the 4-1BB signaling domain includes the nucleic acid sequence described in SEQ ID NO:18, and the CD3ζ signaling domain includes the nucleic acid sequence described in SEQ ID NO:19.

[0153] In one embodiment, the intracellular domain within the CAR of the present invention is designed to include a 4-1BB signaling domain and a CD3ζ signaling domain, wherein the 4-1BB signaling domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:20, and the CD3ζ signaling domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:21.

[0154] In one embodiment, the intracellular domain within the CAR of the present invention is designed to include a 4-1BB signaling domain and a CD3ζ signaling domain, wherein the 4-1BB signaling domain comprises the amino acid sequence described in SEQ ID NO:20, and the CD3ζ signaling domain comprises the amino acid sequence described in SEQ ID NO:21.

[0155] In one embodiment, the anti-CD2 CAR comprises an amino acid sequence described 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 the group consisting of SEQ ID NO: 1 to 7. In one embodiment, the anti-CD5 CAR comprises an amino acid sequence described in any one of SEQ ID NO: 71, 72, 77, 78, 89, or 90. In one embodiment, the anti-CD5 CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8 to 13.

[0156] The acceptable variations in CAR sequences are known to those skilled in the art. For example, in some embodiments, a CAR includes 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 with any of the amino acid sequences described in any of 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 with any of the nucleic acid sequences described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0157] The present invention should be interpreted as comprising any one of the following: a CAR, a nucleic acid encoding a CAR, a vector containing a nucleic acid encoding a CAR, a cell containing a CAR, a cell containing a nucleic acid encoding a CAR, and a cell containing a vector containing a nucleic acid encoding a CAR.

[0158] TIFF2026091929000002.tif108159TIFF2026091929000003.tif223159TIFF2026091929000004.tif219159TIFF2026091929000005.t if223159TIFF2026091929000006.tif225159TIFF2026091929000007.tif227159TIFF2026091929000008.tif218159TIFF20260919290 00009.tif218159TIFF2026091929000010.tif218159TIFF2026091929000011.tif223159TIFF2026091929000012.tif223159TIFF202 6091929000013.tif224159TIFF2026091929000014.tif223159TIFF2026091929000015.tif219159TIFF2026091929000016.tif155158

[0159] CRISPR / CAS In some aspects of the present invention, cells are modified by the CRISPR / Cas system. The CRISPR / Cas system includes, but is not limited to, the CRISPR / Cas9 system and the CRISPR / Cpf1 system. In some aspects, the present invention includes cells modified using the CRISPR / Cas9 system. In some aspects, the modification includes knockout or mutation of an endogenous gene, such as CD2, CD5, or CD7.

[0160] The CRISPR / Cas9 system is a simple and efficient system for inducing target-specific gene alterations. Target recognition by the Cas9 protein requires a conserved trinucleotide containing a "seed" sequence within the guide RNA (gRNA or sgRNA) and a protospacer adjacent motif (PAM) sequence upstream of the gRNA binding region. Therefore, the CRISPR / Cas9 system can be modified to cleave virtually any DNA sequence by redesigning the gRNA for use in cell lines (such as 293T cells), primary cells, and CAR T cells. Because the CRISPR / Cas system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, it is uniquely suited for multiplex gene editing or synergistic activation of target genes.

[0161] CRISPRi, an example of a CRISPR / Cas system used to inhibit gene expression, is described in U.S. Publication No. US2014 / 0068797, which is incorporated herein by reference in its entirety. CRISPRi induces permanent gene disruption by utilizing RNA-induced Cas9 endonuclease to introduce DNA double-strand breaks that trigger the error-prone repair pathway, resulting in frameshift mutations. Cas9 that has lost its catalytic activity lacks endonuclease activity. When co-expressed with guide RNA, a DNA recognition complex is generated that specifically interferes with transcription elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently suppresses the expression of target genes.

[0162] CRISPR / Cas gene disruption occurs when a target gene-specific guide nucleic acid sequence and Cas endonuclease are introduced into a cell, forming a complex that enables the Cas endonuclease to introduce double-strand breaks into the target gene. In one embodiment, the CRISPR system includes, but is not limited to, an expression vector such as the pAd5F35-CRISPR vector. In another embodiment, the Cas expression vector induces the expression of the Cas9 endonuclease. Other endonucleases may 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.

[0163] In one embodiment, induction of a Cas expression vector involves exposing cells to a drug that activates an inducible promoter within the Cas expression vector. In such an embodiment, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., tetracycline or a derivative of tetracycline, e.g., doxycycline). However, it should be understood that other inducible promoters may be used. The inducer may be a selective condition (e.g., exposure to a drug, e.g., an antibiotic) that results in induction of the inducible promoter. This results in the expression of the Cas expression vector.

[0164] The guide nucleic acid sequence is specific to a particular gene and targets that gene for double-strand breaks induced by Cas endonuclease. The sequence of the guide nucleic acid sequence may be located within the gene locus of that 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 nucleotides long, or longer.

[0165] The guide nucleic acid sequence may be specific to any gene, such as CD2, CD5, or CD7. The guide nucleic acid sequence may include RNA sequences, DNA sequences, combinations thereof (RNA-DNA combination sequences), or sequences containing synthetic nucleotides. The guide nucleic acid sequence may be a single molecule or a double molecule. In one embodiment, the guide nucleic acid sequence includes a single guide RNA.

[0166] With respect to the formation of the CRISPR complex, the “target sequence” refers to a sequence designed such that the guide sequence has some complementarity to the target sequence so that hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required, as long as sufficient complementarity exists to induce hybridization and promote the formation of the CRISPR complex. The target sequence may include any polynucleotide, such as a polynucleotide of DNA or RNA. In one embodiment, the target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be located in an organelle of a eukaryotic cell, e.g., a mitochondria or the nucleus. Typically, with respect to the endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence that hybridizes with the target sequence and complexes with one or more Cas proteins) results in a break in one or both strands of the target sequence or near it (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, complete complementarity is not considered necessary, as long as it is sufficient for it to be functional. In one embodiment, when the tracr sequences are optimally aligned, they have at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequences.

[0167] In another embodiment, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into a host cell such that the expression of CRISPR system elements directs the formation of a CRISPR complex at one or more target sites. For example, the Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence may each be functionally linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in a single vector, and one or more additional vectors may provide any components of the CRISPR system not included in the first vector. The CRISPR system elements combined in a single vector may be positioned in any suitable orientation; for example, one element may be positioned 5' ("upstream") or 3' ("downstream") relative to a second element. The coding sequence of one element may be positioned on the same or opposite strand of the coding sequence of the second element, and may be oriented in the same or opposite direction. In one embodiment, a single promoter drives the expression of a transcript encoding a CRISPR enzyme, as well as one or more of the following: a guide sequence, a tracr mate sequence (optionally functionally linked to the guide sequence), and tracr sequences embedded within one or more intron sequences (e.g., each embedded in a different intron, two or more embedded in at least one intron, or all embedded in a single intron).

[0168] In one embodiment, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g., the CRISPR enzyme plus about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains). The CRISPR enzyme fusion protein may contain additional protein sequences and optionally a linker sequence between the two domains. Examples of protein domains that can be fused with a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Additional domains that can form part of a fusion protein containing a CRISPR enzyme are described in US20110059502, which is incorporated herein by reference. In one embodiment, a tagged CRISPR enzyme is used to identify the location of a target sequence.

[0169] Conventional virus-based and non-virus-based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to deliver nucleic acids encoding components of the CRISPR system to cultured cells or host organisms. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery media such as liposomes. Another delivery mode for CRISPR / Cas9 involves a combination of RNA and purified Cas9 protein in the form of a Cas9-guide RNA-ribonucleoprotein (RNP) complex (Lin et al., 2014, ELife 3:e04766). Viral vector delivery systems include DNA viruses and RNA viruses that, after delivery to cells, have either a genome as an episome or an integrated genome (Anderson, 1992, Science 256:808-813; and Yu et al., 1994, Gene Therapy, 1:13-26).

[0170] In one embodiment, CRISPR / Cas is derived from the type II CRISPR / Cas system. In another embodiment, the CRISPR / Cas system is derived from the Cas9 protein. The Cas9 protein may be derived from Streptococcus pyogenes, Streptococcus thermophilus, or other species. In one embodiment, Cas9 may include spCas9, Cpf1, CasY, CasX, or saCas9.

[0171] Generally, CRISPR / Cas proteins contain at least one RNA recognition domain and / or RNA binding domain. The RNA recognition domain and / or RNA binding domain interact with guide RNA. CRISPR / Cas proteins may also contain nuclease domains (i.e., DNase domains or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, and other domains. CRISPR / Cas proteins can be modified to increase nucleic acid binding affinity and / or specificity, alter enzymatic activity, and / or change other properties of the protein. In one embodiment, the CRISPR / Cas-like protein of the fusion protein may be derived from the wild-type Cas9 protein or a fragment thereof. In another embodiment, CRISPR / Cas may be derived from a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein may be modified to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, domains of the Cas9 protein that are not involved in RNA-induced cleavage may be excluded from the protein so that the modified Cas9 protein is smaller than the wild-type Cas9 protein. Generally, the Cas9 protein contains at least two nuclease (i.e., DNase) domains. For example, the Cas9 protein may contain a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains function together to create double-strand breaks in DNA and to cleave single strands (Jinek et al., 2012, Science, 337:816-821). In one embodiment, a Cas9-derived protein may be modified to contain only one functional nuclease domain (either a RuvC-like or HNH-like nuclease domain). For example, a Cas9-derived protein may be modified so that one of the nuclease domains is deleted or mutated and becomes non-functional (i.e., no nuclease activity is present).In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein can introduce nicks into double-stranded nucleic acids (such proteins are called "nickases") but cannot cleave double-stranded DNA. In any of the above embodiments, one or all of the nuclease domains may 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 complete gene synthesis, and other methods known in the art.

[0172] In one non-limiting embodiment, the vector drives the expression of the CRISPR system. A wealth of suitable vectors useful in the present invention are available in the art. The vectors used are suitable for replication and, optionally, for integration in eukaryotic cells. Typical vectors contain transcriptional and translational terminators, start sequences, and promoters useful for controlling the expression of desired nucleic acid sequences. The vectors of the present invention may also be used for standard gene delivery protocols of nucleic acids. Methods of gene delivery are known in the art (U.S. Patents 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety).

[0173] Furthermore, vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4th Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012) and other manuals on virology and molecular biology. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, Sindbisviruses, gamma-retroviruses, and lentiviruses. Generally, a suitable vector contains a functional replication origin, promoter sequence, convenient restriction endonuclease site, and one or more selectable markers in at least one organism (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0174] Nucleic acid introduction 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, and electroporation. RNA can be introduced into target cells using commercially available methods including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.), or Gene Pulser II (BioRad, Denver, Colo.), and Multiporator (Eppendort, Hamburg, Germany). RNA can also be introduced into cells using cationic liposome-mediated transfection with lipofection, polymer encapsulation, peptide-mediated transfection, or particulate gun particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0175] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, are the most widely used methods for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0176] Chemical means for introducing polynucleotides into host cells include colloidal dispersions such as macromolecular complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery medium in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0177] Suitable lipids for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained 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 encompassing various monolayer and multilayer lipid media formed by the formation of sealed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayered liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-reconfiguration before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions with structures different in solution from typical vesicle structures are also included. For example, lipids can take on micelle structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0178] Various assay methods can be performed to confirm the presence of nucleic acids in host cells, regardless of the method used to introduce exogenous nucleic acids into host cells or to expose cells to the inhibitors of the present invention in any other way. Such assay methods include “molecular biological” assay methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; and “biochemical” assay methods, such as detecting the presence or absence of a particular peptide by immunological means (ELISA and Western blotting) or by assay methods described herein for identifying agents that fall within the scope of the present invention.

[0179] Furthermore, nucleic acids can be introduced by any means, such as transduction of enlarged T cells, transfection of enlarged T cells, and electroporation of enlarged T cells. One nucleic acid may be introduced by one method, and another nucleic acid to be introduced into the T cells may be introduced by a different method.

[0180] RNA In one embodiment, the nucleic acid introduced into T cells is RNA. In another embodiment, the RNA is mRNA, including RNA transcribed in vitro or synthetic RNA. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable DNA source. The desired template for in vitro transcription is a chimeric membrane protein. As an example, the template encodes an antibody, a fragment of an antibody, or a portion of an antibody. As another example, the template includes an extracellular domain containing a single-strand variable domain of an antibody, such as anti-CD3, and an intracellular domain of a costimulatory molecule. In one embodiment, the template for the RNA chimeric membrane protein encodes a chimeric membrane protein comprising an extracellular domain containing an antigen-binding domain derived from an antibody against a costimulatory molecule, and an intracellular domain derived from a portion of the intracellular domains of CD28 and 4-1BB.

[0181] PCR can be used to create templates for in vitro mRNA transcription, which are then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have a region substantially complementary to the region of DNA used as the PCR template. As used herein, “substantially complementary” means a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary, or in which one or more bases are non-complementary or mismatched. Substantially complementary sequences can anneal or hybridize with the intended DNA target under the annealing conditions used in PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, a primer can be designed to amplify a portion of a gene that is normally transcribed in cells (the read frame), including the 5' and 3' UTRs. A primer can also be designed to amplify a portion of a gene that codes for a specific domain of interest. In one embodiment, a primer is designed to amplify a coding region of human cDNA that includes all or part of the 5' and 3' UTRs. Primers useful for PCR are prepared by synthetic methods well known in the art. A “forward primer” is a primer that contains a region of nucleotides substantially complementary to the nucleotides on the DNA template upstream of the DNA sequence to be amplified. In this specification, “upstream” refers to the 5’ position of the DNA sequence to be amplified relative to the coding strand. A “reverse primer” is a primer that contains a region of nucleotides substantially complementary to the double-stranded DNA template downstream of the DNA sequence to be amplified. In this specification, “downstream” refers to the 3’ position of the DNA sequence to be amplified relative to the coding strand.

[0182] Chemical structures that have the ability to enhance RNA stability and / or translation efficiency may be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is 0 to 3000 nucleotides long. The lengths of the 5' and 3' UTR sequences appended to the coding region can be varied by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTR. Using this technique, those skilled in the art can vary the lengths of the 5' and 3' UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0183] The 5' and 3' UTRs can be naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, non-endogenous UTR sequences for the gene of interest can be added by incorporating the UTR sequence into forward and reverse primers, or by any other modification of the template. The use of non-endogenous UTR sequences for the gene of interest can be useful in altering RNA stability and / or translation efficiency. For example, AU-rich elements in the 3' UTR sequence are known to reduce mRNA stability. Therefore, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA based on UTR properties known in the art.

[0184] In one embodiment, the 5' UTR may contain the Kozak sequence of an endogenous gene. Alternatively, if a non-endogenous 5' UTR is added to the gene of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. While the Kozak sequence can enhance the translation efficiency of some RNA transcripts, it does not appear to be required for all RNAs to enable efficient translation. The need for Kozak sequences for many mRNAs is well known in the art. In another embodiment, the 5' UTR may be derived from an RNA virus whose RNA genome is stable in cells. In yet another embodiment, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of mRNA.

[0185] To enable RNA synthesis from a DNA template without requiring gene cloning, the transcription promoter should be added upstream of the sequence to be transcribed to the DNA template. When a sequence functioning as an RNA polymerase promoter is added to the 5' end of a forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the reading frame to be transcribed. In one embodiment, the promoter is the T7 polymerase promoter, as described elsewhere in this specification. 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.

[0186] In one embodiment, mRNA has both a 5' end cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and stability of mRNA within the cell. With circular DNA templates, such as plasmid DNA, RNA polymerase produces long chain-like products that are unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end results in normal-sized mRNA that, even after polyadenylation post-transcription, is not effective for eukaryotic transfection.

[0187] On a linear DNA template, phage 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)).

[0188] The conventional method for incorporating polyA / T stretches into DNA templates is molecular cloning. However, incorporating polyA / T sequences into plasmid DNA can cause plasmid instability because plasmid DNA templates obtained from bacterial cells are often highly damaged by deletions and other abnormalities. This makes the cloning procedure not only cumbersome and time-consuming but also often unreliable. This is why a method that allows for the construction of DNA templates with polyA / T 3' stretches without cloning is highly desirable.

[0189] The poly(A) tail of the transcription DNA template can be produced during PCR by using a reverse primer containing a poly(T) tail, such as a 100T tail (size can be 50–5000T), or after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA and reduces RNA 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 consists of 100–5000 adenosine molecules.

[0190] The poly(A) tail of RNA can be further elongated after in vitro transcription using poly(A) polymerase, such as E. coli poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides approximately doubles the translation efficiency of RNA. Furthermore, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase RNA stability.

[0191] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the method 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)).

[0192] RNA produced by the methods disclosed herein may also include an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral sequence, chromosomal sequence, or artificially designed sequence that initiates cap-independent ribosome binding with mRNA and facilitates the initiation of translation. It may also include any solute suitable for cell electroporation, which may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0193] In some embodiments, RNA, such as RNA transcribed in vitro, is electroporated into cells.

[0194] The disclosed methods can be applied to the modulation of T cell activity in basic research and therapy in the fields of cancer, stem cells, acute and chronic infectious diseases, and autoimmune diseases, including the evaluation of the ability of genetically modified T cells to kill target cancer cells.

[0195] This method also offers the ability to control expression levels over a wide range, for example, by varying the amount of promoter or input RNA, allowing for individual regulation of expression levels. Furthermore, PCR-based mRNA production techniques greatly facilitate the design of mRNAs with different structures and combinations of their domains.

[0196] One advantage of the RNA transfection method of the present invention is that RNA transfection is inherently transient and vector-free. The RNA transgene is delivered to lymphocytes as a minimal expression cassette without the need for any additional viral sequences and can be expressed therein after simple in vitro cell activation. Under these conditions, integration of the transgene into the host cell genome is unlikely to occur. Due to the efficiency of RNA transfection and its ability to uniformly modify an entire lymphocyte population, cell cloning is not required.

[0197] Genetic modification of T cells using in vitro transcribed RNA (IVT-RNA) employs two different strategies, both of which are being sequentially tested in various animal models. Cells are transfected with in vitro transcribed RNA via lipofection or electroporation. To achieve long-term expression of the transfected IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications.

[0198] Several IVT vectors are publicly known in the literature and are used in a standardized manner as templates for in vitro transcription, genetically engineered to produce stabilized RNA transcripts. Currently, protocols used in the art are based on plasmid vectors having the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest with an untranslated region (UTR) flanked on either the 3' and / or 5' side, and a 3' polyadenylic cassette containing 50–70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylic cassette by a type II restriction enzyme (recognition sequences corresponding to cleavage sites). Thus, the polyadenylic cassette corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzymatic cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced intracellularly from such constructs.

[0199] RNA offers several advantages over more traditional plasmid or viral methods. Gene expression from RNA sources does not require transcription, and protein products are rapidly produced after transfection. Furthermore, RNA only needs to be accessible to the cytoplasm, rather than the nucleus, thus achieving extremely high transfection rates with typical transfection methods. In addition, plasmid-based methods require the promoter driving the expression of the gene of interest to be active in the cells under study.

[0200] In another context, RNA constructs are delivered to cells by electroporation. See, for example, U.S. Patents 2004 / 0014645, 2005 / 0052630A1, 2005 / 0070841A1, 2004 / 0059285A1, and 2004 / 0092907A1 for formulations and methodologies of electroporation of nucleic acid constructs to mammalian cells. Various parameters, including the electric field strength required for electroporation of any known cell type, are generally known in the relevant research literature and numerous patents and applications in the art. See, for example, U.S. Patents 6,678,556, 7,171,264, and 7,173,116. Apparatus for the therapeutic application of electroporation is commercially available, such as the MedPulser® DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and is described in U.S. 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 cell transfection, as described in U.S. Patent No. 20070128708A1; electroporation can also be used to deliver nucleic acids to cells in vitro; therefore, electroporation-mediated administration of nucleic acids to cells, including expression constructs utilizing any of the many available apparatuses and electroporation systems known to those skilled in the art, represents a remarkable new means of delivering RNA of interest to target cells.

[0201] T cell source In certain embodiments, the source of T cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. Preferably, the subject is human. T cells can be obtained from several 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 may be used. In certain embodiments, T cells can be obtained from units of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll isolation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukocyte-depleting transfusion. Apheresis products typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction, and the cells may be placed in a washing solution that is calcium-deficient and may be magnesium-deficient, or may be many but not all, for a suitable buffer or medium, such as phosphate-buffered saline (PBS), or for subsequent processing steps. After washing, the cells may be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed, and the cells may be resuspended directly in a medium.

[0202] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation through a Percoll® gradient. Alternatively, T cells can be isolated from the umbilical cord. In any case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0203] The umbilical cord blood mononuclear cells isolated in this way can be used to deplete cells expressing specific antigens, including but not limited to CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be achieved using isolated antibodies, antibody-containing biological samples such as ascites fluid, antibodies bound to physical supports, and cell-binding antibodies.

[0204] Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies directed to surface markers specific to negatively selected cells. A preferred method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0205] To isolate a desired cell population by positive or negative selection, the concentrations 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 cell concentration) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In yet another embodiment, more than 100 million cells / ml is used. In yet another embodiment, cell concentrations of 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 50 million cells / ml are used. In yet another embodiment, cell concentrations of 7,500, 8,000, 8,500, 9,000, 95 million, or 100 million cells / ml are used. In yet another embodiment, concentrations of 125 million or 150 million cells / ml can be used. Using high concentrations can lead to increased cell yield, cell activation, and cell growth.

[0206] T cells can also be frozen after the washing step, without requiring a monocyte removal step. While we do not wish 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. Many freezing solutions and parameters are known in the art and useful in this context, but in a non-limiting example, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing medium. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the gas phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as instantaneous uncontrolled freezing at -20°C or in liquid nitrogen, may be used.

[0207] In one embodiment, T cell populations are contained within cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, and T cell lines. In another embodiment, peripheral blood mononuclear cells contain T cell populations. In yet another embodiment, purified T cells contain T cell populations.

[0208] T cell expansion In certain embodiments, T cells disclosed herein can be increased by approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, as well as any and all overall and partial integer multiples between them. In one embodiment, T cells are enlarged in the range of approximately 20-fold to approximately 50-fold.

[0209] After culturing, the T cells are incubated in cell medium within a culture device for a period of time, or until the cells reach a dense or high cell density for optimal subculturing, and then subculturised into another culture device. The culture device may be any culture device commonly used for culturing cells in vitro. Preferably, the density level before subculturing the cells into another culture device is 70% or higher. More preferably, the density level is 90% or higher. The period may be any time suitable for culturing cells in vitro. The T cell medium may be changed at any point during T cell culture. Preferably, the T cell medium is changed approximately every 2-3 days. The T cells are then collected from the culture device and can be used immediately or cryopreserved for later use. In one embodiment, the present invention includes the step of cryopreserving enlarged T cells. The cryopreserved T cells are thawed before nucleic acids are introduced into the T cells.

[0210] In another embodiment, the method comprises the steps of isolating T cells and expanding T cells. In yet another embodiment, the present invention further comprises the step of cryopreserving T cells before expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding chimeric membrane proteins.

[0211] Another procedure for ex vivo cell expansion is described in U.S. Patent No. 5,199,942 (which is incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 may be an alternative option or may be added to other expansion methods described herein. Briefly, ex vivo culture and expansion of T cells involves the addition of cell growth factors, such as those described in U.S. Patent No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, the step of expanding T cells includes the step of culturing T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0212] The culture stages described herein (after contact with the active substance or electroporation as described herein) may be very short, for example, less than 24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culture stages further described herein (after contact with the active substance as described herein) may be longer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.

[0213] Various terms are used to describe cultured cells. Cell culture generally refers to cells taken from an organism and grown under controlled conditions. Primary cell culture is the culture of cells, tissues, or organs taken directly from an organism and prior to the first subculturing. Cells expand in culture, resulting in a larger cell population, when placed in a growth medium under conditions that promote cell proliferation and / or division. When cells expand in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double, also known as the doubling time.

[0214] Each passage in a cell culture is called a passage. When cells are passaged, they are said to have been passaged. A particular cell population or cell line may also be referred to or characterized by the number of passages it has undergone. For example, a cultured cell population that has been passaged 10 times may be called a P10 culture. Primary culture, i.e., the first culture after cell isolation from tissue, is designated as 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 tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that many population doublings can occur during passage; therefore, the number of population doublings in a culture is greater than the number of passages. The expansion of cells during the interpassage period (i.e., the number of population doublings) depends on many factors, including, but not limited to, seeding density, substrate, medium, and interpassage time.

[0215] In one embodiment, cells may be cultured for several hours (approximately 3 hours) to approximately 14 days, or any integer value of time in between. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15, (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum 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 for cell proliferation known to those skilled in the art. Other additives for cell proliferation include, but are not limited to, surfactants, plasmamenates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may be 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, and may be either serum-free or supplemented with an appropriate amount of serum (or plasma) or a specified group of hormones, and / or sufficient amounts of cytokines for T cell proliferation and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in the cultures of cells intended for injection into the target. Target cells are maintained under conditions necessary to support proliferation, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0216] The culture medium used to cultivate T cells may contain agents that can co-stimulate T cells. For example, an agent that can stimulate CD3 is an antibody against CD3, and an agent that can stimulate CD28 is an antibody against CD28. This is because, as demonstrated by the data disclosed herein, cells isolated by the methods disclosed herein can be enlarged by approximately 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x or more. In one embodiment, T cells are enlarged by approximately 20x to approximately 50x or more by culturing a population that has undergone electroporation.

[0217] In one embodiment, a method for expanding T cells may further include a step of isolating the expanded T cells for further application. In another embodiment, a method for expanding T cells may further include subsequent electroporation of the expanded T cells prior to a culture step. Subsequent electroporation may include introducing nucleic acids encoding an active agent into the expanded T cell population, such as transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells, the active agent further stimulating the T cells. The active agent may stimulate the T cells by stimulating further expansion, effector function, or another T cell function.

[0218] Pharmaceutical composition The pharmaceutical compositions of the present invention may, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients, comprise modified cells or modified cell populations as described herein. Such compositions may comprise buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran or mannitol; proteins; amino acids such as polypeptides or 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.

[0219] The pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease being treated (or prevented). The appropriate dosage may be determined by clinical trials, but the amount and frequency of administration will depend on factors such as the patient's condition, as well as the type and severity of the patient's disease.

[0220] The cells of the present invention administered to the patient may be autologous, allogeneic, or heterologous to the patient receiving treatment.

[0221] The cells of the present invention may be administered in doses, routes, and timings determined in appropriate preclinical and clinical experiments and trials. The cell composition may be administered multiple times in doses within these ranges. Administration of the cells of the present invention may be combined with other methods useful for treating a desired disease or condition, as determined by those skilled in the art.

[0222] The pharmaceutical compositions containing modified T cells described herein are 10 4 ~10 9 Cells / kg body weight, in some cases, 10 5 ~10 6It may generally be said that the T cell composition can be administered in doses of cells / kg body weight (including all integer values ​​within these ranges). The T cell composition may be administered multiple times at these doses. The cells may be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment plan for a particular patient can be readily determined by those skilled in the medical field by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0223] The modified cells of the present invention may be administered in any simple manner known to those skilled in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intralymphatically, intramedullarily, intramuscularly, intravenously (iv) or intraperitoneally. In other examples, the cells of the present invention may be injected directly into an inflamed site, a local disease site, lymph nodes, organs, tumors, etc., in a subject.

[0224] It should be understood that the methods and compositions useful in the present invention are not limited to the specific formulations described in the examples. The following examples are provided to those skilled in the art to provide a complete disclosure and description of the present invention regarding methods for preparing and using cells, methods for expanding and culturing cells, and methods for therapeutic use, and are not intended to limit the scope of what the inventors consider to be their invention.

[0225] Unless otherwise indicated, the practical application of the present invention utilizes conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the scope of those skilled in the art. Such techniques are well described 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" and "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); and "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of polynucleotides and polypeptides of the present invention and can therefore be considered in the making and practice of the present invention. Techniques particularly useful for specific embodiments are discussed in the following sections. [Examples]

[0226] Experimental Examples The present invention will be described below 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 become apparent as a result of the teachings provided herein.

[0227] The materials and methods used in these experiments are described below. TIFF2026091929000017.tif34160

[0228] sgRNAs targeting CD2, CD5, or CD7 (e.g., SEQ ID NO: 22-24, respectively) were designed and synthesized using the GeneArt Precision sgRNA Synthesis Kit. Cas9 expression plasmids (pGEM-Cas9) were 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. CD2 / CD5 / CD7 expression in Jurkat cells was detected by flow cytometry to determine the most effective CD2 / CD5 / CD7 sgRNA. Then, CRISPR editing was performed in primary human T cells using the most efficient CD2 / CD5 / CD7 sgRNA. The selected sgRNAs and Cas9 RNA were electroporated into primary human T cells. To validate the knockout / editing efficiency, CD2 / CD5 / CD7 expression in primary human T cells was detected by flow cytometry.

[0229] Specifically, fresh CD4 / CD8 T cells were obtained and incubated with dynabeads on day 0. On day 4, the beads were removed from the cells and electroporated with Cas9 and sgRNA. Conditional medium (TCM (X-vivo15, 5% human serum, glutamine), IL-7 10 ng / ml, and IL-15 10 ng / ml) was added to the cells. On day 6, the cells were transduced with CAR lentivirus. On day 9, CAR expression was assessed. The cells were cultured to 0.8 e6 / ml and frozen when the volume fell below 300 fl (Figure 22).

[0230] CAR constructs: All constructs were generated using the lentivirus pTRPE 4-1BB CD3ζ backbone. The OKT11 CAR and TS2 / 18.1.1 CAR were constructed using scFv derived from antibodies produced from hybridomas purchased from ATCC (ATCC® CRL-8027® and ATCC® HB-195®, respectively). The T11-2 CAR was constructed using scFv derived from antibodies produced from hybridomas received from Ellis Reinherz. All CD5 CARs were constructed using scFv derived from antibody sequences published in WO2010 / 022737 A1, the contents of which are incorporated herein by reference in their entirety.

[0231] The results of the experiment are explained below.

[0232] Example 1: A novel approach to target T-cell lymphoma and T-cell leukemia without causing T-cell damage. T-cell lymphomas and T-cell leukemias generally have a very poor prognosis, and there are few treatment options available for these patients. Chimeric antigen receptor T-cell (CART) immunotherapy has yielded unprecedented results in CD19+ B-cell non-Hodgkin lymphoma (B-NHL). Herein, another successful “CART19-like” product was designed to target T-NHL. Since CD19 is not expressed in T-NHL, additional targets such as CD2, CD5, CD7, and others were evaluated for CART 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 invention, a safe and effective CART strategy for treating T-cell lymphoma was developed by editing normal T cells to be resistant to CART-induced death (Figure 2). When CART targets (CD2, CD5, CD7) are temporarily removed from normal T cells, thereby avoiding CART-mediated cell death and immunodeficiency, CART therapy for T-cell lymphoma and T-cell leukemia becomes feasible (Figure 2).

[0233] Example 2: Anti-CD5 CAR T cells (CART5) and CD5 knockout (KO) normal T cells A dual immunotherapy involving anti-CD5 CAR T cells (CART5) and CD5 knockout (KO) normal T cells is disclosed herein (Figure 3). CART5 destroys T-cell lymphoma (e.g., T-NHL) cells or T-cell leukemia cells, but also kills normal T cells. Infusion of CD5 KO normal T cells provides CART-resistant T-cell immunity until the CART5 cells are depleted, using a suicide gene (e.g., iCasp9, CD20 / rituximab, or others) in some cases.

[0234] CD5 was selected as a T-NHL target because it is highly expressed in T-NHL cells and not expressed in other tissues other than T cells and a small subset of B cells. Six anti-CD5 CAR constructs were generated using single-chain variable fragments (scFv) with different affinities (#17, #34, and #9, respectively, with high, medium, and low affinity (Klitgaard JL, et al. (2013) British Journal of Haematology 163:182-93)) and expressed in T cells (Figures 4-5). Interestingly, although CD5 expression was lower compared to control T cells (Figure 8), anti-CD5 CART did not require CD5 knockout to produce CART5 cells, even though CD5 was expressed in 100% of CART cells (Mamonkin M, et al. (2015) Blood 126:983-92). Without CRISPR-Cas9 knockout of CD5, the mean fluorescence intensity (MFI) of CD5 was 10 times lower in CART5 compared to control T cells, but there was no change in other pan-T cell markers such as CD2 (Figure 8).

[0235] The in vitro and in vivo activity of different CART5 constructs was compared. Construct C3054, derived from high-affinity scFv#17, showed the best in vivo cell death. Jurkat cells were transduced with different CAR5 constructs (Figure 13, targeted epitopes and affinities shown on the left) and a GFP-NFAT reporter, and then co-cultured with CD5+ tumor cells (or controls) for 24 hours. Lead CART5 (C3054) showed increased NFAT activation (Figure 13).

[0236] We implemented a lead anti-CD5 CART using a suicide system and tested its function in vitro and in vivo. While we do not wish to be constrained by any particular theory, we suggest that CD5 removal (CRISPR-Cas9 KO) further enhances the antitumor effect of CART5 by eliminating the possibility of cis-surface interactions between CART5 and CD5 on CART5.

[0237] Insertion of suicide pathway into lead CART5 product: The lead candidate CART5 (C3054) product was modified to express a suicide pathway (Figure 26). A P2A 2 cistron vector (Di Stasi A, et al. (2011) 365:1673-83) (iCART5) encoding both CAR5 and the inducible caspase 9 suicide pathway was developed. To define the most efficient, both directions, CAR5-P2A-iCasp9 and iCasp9-P2A-CAR5 (Figure 26) were cloned (for safety, higher % dual-expression cells and lower % CAR5+iCasp9-). iCART5 was efficiently eliminated using the clinical-grade compound limituside (AP1903, Bellicum Pharmaceuticals).

[0238] In vitro testing of iCART5: Newly generated iCART5 will be compared to WT CART5 to confirm efficacy (in vitro luciferase-based cell death) and phenotype / function (flow cytometry phenotype, 30-plex cytokine analysis by Luminex assay, CFSE proliferation, and CD107a degranulation) upon antigen stimulation (CD5+ Jurkat T leukemia cells). Importantly, in vitro depletion will be tested by co-culturing iCART5 with different concentrations of limituside (0, 0.03, 0.3, 3, 10 nM) and checking for cell death at 15 min, 30 min, and 2, 6, 12, and 24 hours. iCART5 will also be tested against primary T-NHL cells using an established cell death assay with primary CFSE-labeled Sezary cells.

[0239] In vivo trial of iCART5: An in vivo xenograft model (Ruella M, et al. (2016) J Clin Invest) using Click Beetle Green (CBG) + Jurkat T leukemia cell line and Click Beetle Red (CBR) + iCART5 will be used to test the ability of limitudide (50 ug / 23 mice) to deplete iCART5 versus WT CART5 in vivo. NOD SCIDγ-deficient mice (NSG) (8 mice per group) will receive 2 × 10 e 6 CART5 cells / mice are injected. Tumor burden is assessed over time as bioluminescence (CBG), T cell phenotype is studied by flow cytometry at multiple time points (hours / days), and expansion is studied by bioluminescence (CBR). Mice are maintained for a long period (3-4 months) for monitoring of survival and recurrence. A human T-NHL xenograft model has been previously established by IV injection of primary Sezary cells. This model is used to test iCART5 for both antitumor and depletion efficiency.

[0240] Evaluation of the role of CD5 KO in CART5: Preliminary data demonstrated that CD5 CART5 is more effective than WT CART5 in vivo. CD5 is knocked out in CART5 cells using CRISPR-Cas9. The CRISPR-Cas9 CART expansion protocol has been previously optimized. Wild-type CART5 is compared to CD5 KO CART5 in vitro by examining CART5 viability, antigen-driven proliferation (using CFSE labeling), cytokine production (by 30plex Luminex), degranulation (CD107a assessment by flow cytometry), cytotoxicity (luciferase-based), and phenotype (memory subset, Th1 / Th2). Both cell lines (e.g., Jurkat) and primary samples are used as targets. We will perform an in vivo comparison of CD5 KO versus WT CART5 (1 × 10 e6 cells / mouse) in Jurkat-carrying NSG mice by monitoring peripheral blood enlargement and phenotype on days 10 and 14.

[0241] Mechanism of CART5 function enhancement by CD5 KO: Since CD5 KO has been shown to improve CART5 activity, additional studies will be 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) to demonstrate that CAR5 binds to CD5 in cis; (iii) CAR5 expression in CD5+Jurkat to show that CART5 cannot kill CAR5+Jurkat because the CD5 epitope is masked (by CAR5); and (iv) studying CART5 activation in the presence or absence of CD5, as CD5 plays an inhibitory role in T cell activation (phosphoflow cytometry).

[0242] Generation of CART-resistant normal T cells to avoid T cell malformation: Since both tumor T cells and normal T cells express similar levels of CD5, CART5 cannot distinguish them. To ensure immunity during CART antitumor activity, we developed CART-resistant normal T cells for co-injection with anti-T-NHL CART. CRISPR-Cas9 gene editing was used to knock out CD5 in normal T cells, thereby making them invisible to CART5. Using an optimized CART expansion protocol, a highly efficient CRISPR-Cas9 gRNA (#4) capable of knocking out approximately 95% of CD5 in normal T cells was generated. Data showed that CD5 KO T cells were resistant to CART5, while WT T cells (CD5+) were potently killed within 24 hours.

[0243] Production of CD5 KO Normal T Cells: CD5 KO Normal T cells are developed using a highly efficient gRNA (#4) that is electroporated with the Cas9 protein (ThermoFisher v2) using a Lonza 4D Nucleofector. CRISPR-Cas9 KO is performed on day 1, and the cells are then cultured at 30°C for 2 days to increase gene editing, and then activated with anti-CD3 / CD28 Dynabeads (Beads::1 T cells) and expanded to a cell volume of less than 300 fl.

[0244] In vitro evaluation of resistance of CD5 KO normal T cells to iCART5-mediated death: Resistance of CD5 KO normal T cells to CART5 is tested in vitro by performing a death assay (CFSE labeling of target T cells). Preliminary results showed that CD5 KO confers resistance. Three additional T cell donors are tested.

[0245] In vivo evaluation of resistance of CD5 KO normal T cells to iCART5 in an autologous xenograft model: NSG mice (8 mice / group) are transplanted with luciferase-+CD5 KO normal T cells or WT cells, and autologous iCART5 is injected 2 days later. The effect of iCART5 on CD5 KO normal T cells and WT normal T cells is assessed by bioluminescence. Once WT T cells are completely eliminated by iCART5 (luminescence), limituside is administered to deplete iCART5. Then, WT T cells are reinjected to demonstrate that normal T cells can regrow in the host. Blood is drawn from the mice weekly to assess the expansion of CART.

[0246] Evaluation of the role of CD5 KO on normal T cell function: The role of CD5 KO in normal T cells will be investigated by carefully studying T cell effector function. Following TCR-specific stimulation (anti-CD3 / CD28 beads), cytokine production (30-plex Luminex), proliferation (CFSE), and activation will be measured for CD5 KO T cells versus WT cells. We will also test whether CD5 KO T cells proliferate and produce cytokines similarly to WT cells when exposed to common infections.

[0247] Defining the optimal CD5KO normal T cell dose for clinical use: Using both in silico and experimental approaches (TCR sequencing and tetramer staining of TCRs specific to infectious pathogens), define the minimum number of cells to be injected into relapsed or refractory (r / r) T-NHL patients to ensure sufficient T cell immunity against the most common infections.

[0248] A Phase 1 pilot clinical trial of co-infusion of iCART5 and CD5 knockout normal T cells in patients with advanced T-cell lymphoma: Develop and submit an Investigation New Drug (IND) package to the FDA. Initiate a Phase 1 clinical trial to test an anti-T-NHL CART approach in patients. The IND package will be based on preliminary results and further data from the experiments described herein.

[0249] Clinical Trial Protocol Design: Phase 1 clinical trials will include patients with r / r T-NHL treated using a 3+3 protocol design. From a single apheresis, enriched T cells will be used to produce two products: #1. CRISPR-Cas9 CD5 KO normal T cells and #2. iCART5. The first product to be injected will be KO normal T cells, followed the next day by the injection of iCART5. The first cohort of patients will receive lymphocyte depletion [cyclophosphamide (60 mg / kg × 2 days) and fludarabine (25 mg / m2 × 5 days)] as well as product #1. If no dose-limiting toxicity (DLT) is observed, cohort 2 will receive lymphocyte depletion, product #1, and 1–5 × 10⁻⁶ e⁻¹ over 3 days. 7 It accepts all iCART5 (product #2) (10%, 30%, and 60% of the total dose). 1~5 × 10 e 7 The total CART5 dose is below optimal based on the CART19 experiment in B-NHL8. If DLT is not observed in Cohort 2, Cohort 3 is lymphocyte depletion, product #1, and 1-5 × 10 e 8 Patients will receive the full dose of CART5. Patients from Cohort #1 will be permitted to proceed to Cohort #2 if no toxicity is observed within the first four weeks. To prevent potential long-term T-cell damage, iCART5 cells will be depleted using the dimerizing agent limituside (NCT02744287), based on tumor clearance (and maximum value at 6 months).

[0250] Preparation and submission of IND packages to the FDA: Optimize clinical-grade manufacturing in collaboration with the Clinical Vaccine and Cell Production Facility (CVPF). Prepare all documented preclinical trial results, along with the clinical trial protocol, in a format suitable for IND submission. Extensive support for IND preparation is available at CCI and ACC.

[0251] Patient enrollment and treatment: Following successful submission of the IND and approval from all regulatory authorities, a Phase 1 trial will be initiated at the University of Pennsylvania's Lymphoma Program (Director: Dr. Stephen Schuster; Scientific Director: Dr. Marco Ruella). The Lymphoma Program has a dedicated Clinical Research Unit (CRU) with extensive experience in early-stage research management. Dr. Ruella is the Principal Investigator for this trial, and Dr. Carl June is the Scientific Protocol Advisor. Manufacturing of the two products will be carried out at CVPF.

[0252] Correlative studies: Patient samples (peripheral blood) will be analyzed at multiple time points (apheresis, -1, 0, 7, 14, 28, 60, 90 days) to test CART expansion (qPCR and flow cytometry), CART phenotype (CyTOF), CART gene expression profiling (GEP) (NanoString, single-cell RNA-seq, 10X Genomics), and serum cytokine levels (Luminex, 30-plex array). Additional studies will be conducted on pre- and post-treatment tumor biopsy materials when available (RNA-seq and Hyperion analysis of the tumor microenvironment).

[0253] This trial will represent a significant milestone in the development of novel combination immunotherapies, as it represents an innovative immunotherapeutic approach to treat T-cell non-Hodgkin lymphoma while avoiding 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 involves the co-infusion of normal T cells with CD5 knocked out, thereby ensuring immunological protection by T cells during CART5 antitumor activity. Subsequently, to ensure long-term normal immunological reconstitution, the CART5 cells are depleted using a suicide system. This is one of the first CART trials for T-NHL and the only trial that includes a two-pronged approach to address the toxicity issue. T-NHL has an extremely poor prognosis, and there are currently no active immunotherapies available. Therefore, the development of such an innovative strategy represents a vertical advance in the fields of hematology and immunotherapy. Based on the clinical results of Phase 1 trials and findings from correlational studies, this strategy can be implemented to target multiple targets simultaneously to avoid antigen loss escape (e.g., CART5 + CART7), or CART can be combined with small molecules that can enhance CART-mediated death.

[0254] Example 3: Anti-CD2 CAR T cells (CART2) and CD2 knockout (KO) normal T cells A dual immunotherapy approach involving anti-CD2 CAR T cells (CART2) and CD2 knockout (KO) normal T cells is disclosed herein (Figure 3). CART2 destroys T-cell lymphoma (e.g., 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 the CART2 cells are depleted, in some cases by using a suicide gene (e.g., iCasp9).

[0255] Guide RNAs were designed to knock out the CD2 gene (and CD5) using the CRISPR / Cas9 system. CD2 was effectively knocked out in 78% of the T cell population. Second-generation anti-CD2 CARs and anti-CD5 CARs (CART2 and CART5, respectively) were generated (Figure 4). Knockout cells (CD2KO and CD5KO) were incubated with corresponding CART cells (CART2 and CART5, respectively), stimulated, and population doubling was measured (Figure 7). Mock electroporated cells without gRNA were used as a control for comparison. Without CD2 KO, CART2 cells did not expand (Figure 7). With KO, CART2 and CART5 reached population doubling of approximately 5–8 (Figure 7).

[0256] Jurkat cells were transduced with different CAR2 constructs and a GFP-NFAT reporter, and then co-cultured with CD2+ tumor cells (or controls) for 24 hours. Lead CART2 (C3043) showed increased NFAT activation (Figure 14).

[0257] The CART expansion protocol was optimized. CART2 and CART5 cells continued to expand for up to 18 days when incubated with CD2 KO cells or CD5 KO cells (Figure 9).

[0258] Example 4: Testing of CART2 and CART5 Figure 6 shows the CD5 (or CD2 or CD7) KO manufacturing process and CRISPR-Cas9 KO efficiency.

[0259] Six different CAR2 constructs and six CAR5 constructs were challenged in vitro by co-culture with luciferase+Jurkat cells (T cell leukemia cell line). At 24 hours, total killing was measured as the relative decrease 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). Lead CART candidates (CART2 C3043 and CART5 C3054) were selected and tested. The effect of CD2 or CD5 knockout on CART function was tested. T cells resistant to CART2 and CART5 were successfully generated (CD5 and CD2 were knocked out in normal T cells).

[0260] The activities of CART2 and CART5 against cutaneous T cell lymphoma were tested. A 24-hour killing assay was performed. CART2 cells were active against primary Sézary cells (leukemic cutaneous T cell lymphoma) and HH Sézary cell line (Figure 15). CART5 was also active against HH cells (Figure 15).

[0261] The in vivo efficacy of CART2 and CART5 was measured. NSG mice were transplanted with luciferase+Jurkat cells and on day 7, the mice were randomized to receive either control T cells or CART2 or CART5 (1×10 6 ). Mice were imaged weekly using an IVIS Xenogen Spectrum and analyzed by LivingImage software. CART2 C3043 and CART5 C3054 were the most effective (Figure 12).

[0262] CART2 and CART5 were shown to recognize and kill normal T cells (autologous and allogeneic) (Figure 16).

[0263] The removal of CAR targets was also proven to protect normal T cells from CART - induced killing (Figure 17). CD5 KO T cells were resistant to killing by CART5, while WT normal T cells were not. 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 killing by CART2 or CART5, respectively (Figure 17).

[0264] CMV - specific T cells were present in 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 dextramer (SEQ ID NO:101); ICS 4 hours after exposure to CETF peptide; after secondary culture with CMV - peptide - pulsed APCs).

[0265] Example 5: Anti-CD7 CAR T cells (CART7) and CD7 knockout (KO) normal T cells A two - pronged immunotherapy approach involving anti - CD7 CAR T cells (CART7) and CD7 knockout (KO) normal T cells is disclosed herein. CART7 destroys T - cell lymphoma (e.g., T - NHL) or T - cell leukemia cells, but also kills normal T cells. Injection of CD7 KO normal T cells provides CART - resistant T - cell immunity in some cases until CART7 cells are depleted, for example, by using a suicide gene (e.g., iCasp9).

[0266] Guide RNAs were designed to knock out the CD7 gene using the CRISPR / Cas9 system. CD7 was effectively knocked out in 79% of the T - cell population (Figure 25). Six anti - CD7 CARs were generated (Figure 25).

[0267] Example 6: Bispecific CAR T cells Two lentiviral constructs containing CAR5 (C3054) and CAR2 (C3043) linked by a P2A sequence were generated (Figure 19). Gene expression was driven by the EF1α promoter. The CAR5 construct possesses a 4-1BB costimulatory domain and a CD3ζ signaling domain. Efficient knockout of both CD2 and CD5 in normal T cells was demonstrated (Figures 20A-20B).

[0268] Example 6: CD5 knockout enhances CART immunotherapy. CD5 KO CART5 was proven to be more effective than CD5 + CART5 in vivo. CD5 KO increased the antitumor effect of CART5 (Figure 21). In a Jurkat T-ALL xenograft model using NSG mice, CD5 KO CART5 (2 × 10⁶) 6 Cells / mouse yielded a complete long-term response and longer survival compared to WT CART5 (Figure 21).

[0269] CD5 knockout CART19 was also more effective than CD5 + CART19 in vivo. CD5 knockout increased the antitumor efficacy of CART19 (Figure 22). In a NALM6 B-ALL xenograft model, CD5 knockout CART19 showed significantly better tumor control compared to WT CART19 (Figure 22).

[0270] CART5 and CART2 were also able to target 20% of AML cells. CART2 cells were co-cultured with CD2+ AML cells and showed significant cell death at 24 hours (Figures 23A-23B).

[0271] CART5 also targeted 100% of CLL and MCL cells. Cytotoxicity assays demonstrated that CART5 cells could recognize and kill CD5+ MCL cell lines (Jeko-1 and Mino) (Figure 24).

[0272] These data demonstrate that CART therapy is enhanced when treated with anti-CD5 CARs by knocking out CD5, or, surprisingly, when treated with different CAR T cells (e.g., CD19 CART cells).

[0273] Other embodiments Any description of a variable in this specification that includes a list of elements in any definition of a variable includes the definition of that variable as any single element or combination (or partial combination) of the listed elements. Descriptions of embodiments in this specification include that embodiment as a single embodiment, or that embodiment in combination with any other embodiment or a part thereof.

[0274] All patents, patent applications, and publications disclosed herein by reference are incorporated herein by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims shall be construed to include all such embodiments and equivalent variations.

[0275] Sequence information SEQUENCE LISTING <110> The Trustees of the University of Pennsylvania <120> Use of CD2 / 5 / 7 Knock-Out Anti-CD2 / 5 / 7 Chimeric Antigen Receptor T cells Against T Cell Lymphomas and Leukemias <150> US 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 cagtgtata 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 cttttggtcaa 720 gggacgaaac tggagatcaa atccgga 747 <210> 2 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> CD2-OCT11H2L-3029 <400> 2 ggatcccaag ttcagcttca gcaaccaggt gctgaattgg tccgccctgg aactagcgtt 60 aaactgtctt gtaaggcatc cggttatacg tttacaagtt attggatgca ctggattaag 120 caaaggcccg aaaggcct 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 540. tgccgctctt ctaaaaccct cttgcattct aacggcaata catatttgta ttggttcctt 600. 600. gcaagtgctt attattagga tgtctaactt ggctagtggg gtgccaaata ggttcagtgg gtctggatct gagacaactt tcacgttgag aataagtagg gtggaagctg aagacgtcgg fathers tgtatgcagc atttggagta cccttacact ttcgggggag gtactaagct cgaattaaa tccgga 756 <210> 3 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> CD2-OKT11L2H-3030 CAR <400> 3 60. ggatccgata tagttatgac gcaagcagca ccctctgtac ctgtgacacc gggtgaatcc gttagtatct catgccgctc ttctaaaacc ctcttgcatt ctaacggcaa tacatatttg tattggttcc ttcaacgacc aggacaatca ccgcaagtgc ttatttatag gatgtctac ttggctagtg gggtgccaaa taggttcagt gggtctggat ctgagacaac tttcacgttg agaataagta gggtggaagc tgaagacgtc ggtatatact actgtatgca gcatttggag 300 tacccttaca ctttcgggg 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 gaaaattta 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 aaacttagtt gtaaagctag tggtacact tttacgt tctggatga tgggtgaag 120 caacgaccag gccaaggtct ggaatgatc ggcatgattg acccgtctga ctcagaagct 180 cattacaacc agatgttca ggacaagcg actctgactg ttgataaag ctcaagcacc 240 gcctacatgc agctcagtag cctcacatcc gaggattccg cagtgtacta tgcgcgagg 300 ggacgagggt atgatgacgg cgatgcgatg gactattggg gandagggac cagcgtaaca 360 gtcagtagtg gtggcggagg gagcggcggt ggaggaagcg gaggcggagg ttccgatata 420 gttatgaccc agtctcccgc ctctctggcc gttagcttgg vakaacgcgc taccactct 480 taccgagcgt ctaagtccgt cagtacaagc ggttatagtt acatgcactg gaaccagcaa 540 aagcccggac aacctccgag actcctgatt tattttctct ctaccttga gtcaggtgtc 600 ccagccagat tctccggctc tggaagcggc actgacttta cattgaacat tcaccccgtg 660 gaggaggaag acgctgctac ctactattgc atgcaatca cgcactatcc ctacacattc 720 ggggggggca cgaattgga aatcaaatcc gga 753 <210> 5 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> CD2-TS2-18.1.1-H2L-3032 CAR <400> 5 ggaccgagg ttcagcttga ggagagtggg ggaggttttgg taatgccagg tgggtctttg 60 aaactcagtt gcgcggcgtc aggcttcgca ttttcctcct acgatatgtc ctgggtcaga 120 cagacaccg agaagcggct ggaatgggtc gcttacattt ccgggggagg attcacgtac 180 taccggata 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 tctgagctg 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 gttactcac 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 agattacgttt gagaagttca aaaagaaagt cacacttact 600 gcggacacat ctagtagcac cgcatatatg gaactgagca gtctcacctc agacgacacc 660 gcagtgtact attgcgctcg cggaaagtttt 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 aatacgc agaaattcaa ggggaaggcc acctgaccg 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 ctacacttc 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 gacacaaaaat acgcgcagaa attcaagggg aaggccaccc tgaccgtaga taaagctagt 600 tctactgcgt acttgcaact gaaaagcctc acttctgagg actctgccgt ctactactgt 660 gctcggcgaa tgataacgac gggggactgg tatttcgatg tttggggtac aggactacg 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 ataaagatt actacattca ctgggtcaaa 120 cagcgccgg agcaggggct tgaatggatc gggtggattg atcctgaaaa cgggcgcacc 180 gaatatgctc ccaagttcca gggcaaagct actatgaccg ctgacacctc tagtaacact 240 gcctacctgc agttgagctc tcttacgtct gaggataccg ctgtgtacta ctgtaataac 300 ggaaattatg tacgacacta ttacttcgac tactggggc 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 ccgccccggtt tagtggctcc 600 ggttctggga caagttactc cctcacgatc agcagggttg aagccgagga cgctgccact 660 tactattgcc aacagtggtc aagtaaccc aggactttcg gggagaggaac 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 ttcggggga 300 ggaactaaac ttgaaatcaa aggtggcgga gggagcggcg gtggaggaag cggaggcgga 360 ggttccgagg ttaaactcgt ggagagcggt gccgaactcg tccgaagtgg tgcttccgtt 420 aaactcagtt gtgccgcgtc agatttaac 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 tttttacga 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 cgggcattc 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 aggagatgg ctgtagctgc cgatttccag agaagaga aggagatgt 120 failure 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 acgagagag gagtacgatg tttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagatagat ggcggaggcc tacagtgaga ttgggatga aggcgagcgc 240 cggaggggca aggggcacga tggctttac cagggtctca gtacagccac cagggacacc 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 Leu Leu Tyr Ile Phe 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> Artificial 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> Artificial 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> dextramer <400> 101 Asn Leu Val Pro Met Val Ala Thr Val 1 5

Claims

1. A method for treating cancer in a subject requiring it, including the following steps: A step of administering a first modified cell containing a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain to the subject, and A step of administering a second modified cell in which the endogenous CD5 gene is knocked out to the subject.

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

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

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

23.

5. The antigen-binding domain of CAR supports CD5, CD19, CD2, CD7, tumor-specific antigen (TSA), tumor-associated antigen (TAA), glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, and mut. hsp70-2, M-CSF, prostase, 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, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, MART-1 / Melan A (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBV A, HPV antigen E6, HPV antigen E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p The method according to any one of the claims, which can bind to an antigen selected from the group consisting of 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, 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\cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

6. The method according to any one of the claims, wherein the antigen-binding domain of the CAR includes a complementarity-determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31-36, 43-48, 53-58, 65-70, 83-88, and 95-100.

7. The method according to any one of the claims, wherein the antigen-binding domain of the CAR includes a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 41, 51, 63, 75, 81, and 93.

8. The method according to any one of the claims, wherein the antigen-binding domain of the CAR includes a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 42, 52, 64, 76, 82, and 94.

9. The method according to any one of the claims, wherein the antigen-binding domain of the CAR comprises an scFv having an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79, 80, 91, and 92.

10. The method according to any one of the claims, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 37, 38, 49, 59, 60, 71, 72, 77, 78, 89, and 90.

11. The method according to any one of the claims, wherein the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 13.

12. The method according to any one of the claims, wherein CAR further comprises a suicide gene.

13. The method according to claim 12, wherein the suicide gene is iCaspase9.

14. The method according to any one of the claims, wherein the first and second modified cells are T cells.

15. The method according to any one of the claims, wherein the cancer includes T-cell lymphoma or T-cell leukemia.

16. The method according to any one of the claims, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

17. A method for treating cancer in a subject requiring it, including the following steps: A step of administering a first modified cell containing a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain capable of binding to CD2, to the subject, and A step of administering a second modified cell in which the endogenous CD2 gene is knocked out to the subject.

18. The method according to claim 17, wherein the antigen-binding domain of the CAR includes a complementarity-determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31-36, 43-48, 53-58, and 65-70.

19. The method according to claim 18, wherein the antigen-binding domain of the CAR includes a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 41, 51, and 63.

20. The method according to any one of claims 17 to 19, wherein the antigen-binding domain of the CAR includes a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 42, 52, and 64.

21. The method according to claim 18, wherein the antigen-binding domain of the CAR comprises an scFv having an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 28, 39, 40, 50, 61, and 62.

22. The method according to claim 18, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 37, 38, 49, 59, and 60.

23. The method according to claim 18, wherein the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 7.

24. A method for treating cancer in a subject requiring it, including the following steps: A step of administering a first modified cell containing a CAR having an antigen-binding domain, a transmembrane domain, and an intracellular domain capable of binding to CD5 to the subject, and A step of administering a second modified cell in which the endogenous CD5 gene is knocked out to the subject.

25. The method according to claim 24, wherein the antigen-binding domain of the CAR includes a complementation-determining region (CDR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 83-88 and 95-100.

26. The method according to claim 24, wherein the antigen-binding domain of the CAR includes a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 75, 81, and 93.

27. The method according to claim 24, wherein the antigen-binding domain of the CAR includes a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 76, 82, and 94.

28. The method according to claim 24, wherein the antigen-binding domain of the CAR comprises an scFv containing an amino acid sequence selected from the group consisting of SEQ ID NO: 73, 74, 79, 80, 91, and 92.

29. The method according to claim 24, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 71, 72, 77, 78, 89, and 90.

30. The method according to claim 18, wherein the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8 to 13.

31. A method for treating cancer in a subject requiring it, including the following steps: A step of administering a first modified cell containing a CAR having an antigen-binding domain, a transmembrane domain, and an intracellular domain capable of binding to CD7 to the subject, and A step of administering a second modified cell in which the endogenous CD7 gene is knocked out to the subject.

32. The method according to any one of claims 17 to 31, wherein the first and second modified cells are T cells.

33. The method according to any one of claims 17 to 32, wherein the cancer includes T-cell lymphoma or T-cell leukemia.

34. The method according to any one of claims 17 to 32, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

35. The method according to any one of claims 17 to 34, wherein an endogenous gene is knocked out using the CRISPR / Cas9 method.

36. The method according to claim 35, wherein the CRISPR / Cas9 method utilizes an sgRNA containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 22 to 24.

37. The method according to any one of claims 17 to 36, wherein CAR further comprises a suicide gene.

38. The method according to claim 37, wherein the suicide gene is iCaspase9.

39. A nucleic acid containing a CAR, which includes an antigen-binding domain, a transmembrane domain, and an intracellular domain capable of binding to CD2.

40. The nucleic acid according to claim 39, wherein the antigen-binding domain comprises a complementarity-determining region (CDR) containing an amino acid sequence selected from the group consisting of SEQ ID NO: 31-36, 43-48, 53-58, and 65-70.

41. The nucleic acid according to claim 39, wherein the antigen-binding domain comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 41, 51, and 63.

42. The nucleic acid according to claim 39, wherein the antigen-binding domain comprises a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 30, 42, 52, and 64.

43. The nucleic acid according to claim 39, wherein the antigen-binding domain of the CAR comprises an scFv containing an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 28, 39, 40, 50, 61, and 62.

44. The nucleic acid according to claim 39, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 37, 38, 49, 59, and 60.

45. The nucleic acid according to claim 39, wherein the CAR is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 7.

46. A nucleic acid containing a CAR, which includes an antigen-binding domain, a transmembrane domain, and an intracellular domain capable of binding to CD5.

47. The nucleic acid according to claim 46, wherein the antigen-binding domain comprises a complementarity-determining region (CDR) containing an amino acid sequence selected from the group consisting of SEQ ID NO: 83-88 and 95-100.

48. The nucleic acid according to claim 46, wherein the antigen-binding domain comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 75, 81, and 93.

49. The nucleic acid according to claim 46, wherein the antigen-binding domain comprises a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 76, 82, and 94.

50. The nucleic acid according to claim 46, wherein the antigen-binding domain of the CAR comprises an scFv containing an amino acid sequence selected from the group consisting of SEQ ID NO: 73, 74, 79, 80, 91, and 92.

51. The nucleic acid according to claim 46, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 71, 72, 77, 78, 89, and 90.

52. The nucleic acid according to claim 46, wherein the CAR is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 8 to 13.

53. A vector comprising the nucleic acid according to any one of claims 39 to 52.

54. A cell comprising the nucleic acid according to any one of claims 39 to 52 or the vector according to claim 53.

55. A composition comprising a first modified cell containing a chimeric antigen receptor (CAR) comprising a CD2-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD2 gene is knocked out.

56. A composition comprising a first modified cell containing a chimeric antigen receptor (CAR) comprising a CD5-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD5 gene is knocked out.

57. A composition comprising a first modified cell containing a chimeric antigen receptor (CAR) comprising a CD7-targeting antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD7 gene is knocked out.

58. A composition comprising a first modified cell containing a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular domain, and a second modified cell in which the endogenous CD5 gene is knocked out.

59. The antigen-binding domain supports CD5, CD19, CD2, CD7, tumor-specific antigen (TSA), tumor-associated antigen (TAA), glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, and mut. hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostain, PSMA, Her2 / neu, Survivin, telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, MART-1 / Melan A (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBV A, HPV antigen E6, HPV antigen E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p The composition according to claim 58, which can bind to an antigen selected from the group consisting of 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, 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\cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

60. The composition according to any one of claims 55 to 59 and a pharmaceutically acceptable carrier.

61. The composition according to any one of claims 55 to 60, wherein the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 13.

62. The composition according to any one of claims 55 to 60, wherein CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 37, 38, 49, 59, 60, 71, 72, 77, 78, 89, and 90.

Images