Use of CD2 / 5 / 7 knock-out anti-CD2 / 5 / 7 chimeric antigen receptor t cells against t cell lymphomas and leukemias

Abstract

20 26 22 32 98 27 A ug 2 02 6 A B S T R A C T 2 0 2 6 2 2 3 2 9 8 2 7 2 0 2 6 A u g

Description

This disclosure describes three chimeric antigen receptors (CARs) that target T cell neoplasms as well as a method to prevent fratricide of healthy T cells. T cell lymphomas and 30 leukemias are aggressive neoplasms derived from T cell progenitors or differentiated T cells. Mature or peripheral T-cell lymphomas account for 10%-15% of all non-Hodgkin's 2026223298   27 Aug 2026 lymphomas, or -7,000-10,000 cases in the U.S / yr. T cell lymphomas and leukemias have poor prognoses and there are few available treatments. CART therapy has demonstrated efficacy for B-cell neoplasms, but until now, extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies has been problematic because most target 5 antigens are shared between normal and malignant cells, leading to CAR T cell fratricide. Herein CRISPR-Cas editing is used to remove the target antigen from healthy T cells, protecting them from CART cell therapy and eliminating the potentially fatal immunosuppression that would result from elimination of the T-cell compartment. In certain embodiments, the CARs target the T cell antigens CD2, CD5 and CD7. In 10 certain embodiments, CRISPR-Cas knock-out of the CD2, CD5, or CD7 target in healthy T cells prevents the killing of healthy T cells during manufacturing and subsequent CART therapy. Methods of Treatment 15          The present invention includes a method for treating a T cell lymphoma or T cell leukemia in a subject in need thereof. In another aspect, the invention includes a method for preventing CAR T cell fratricide in a subject in need thereof. In certain embodiments, the method comprises administering to the subject a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an 20 antigen binding domain that targets CD2, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell wherein the endogenous CD2 gene has been knocked-out. In certain embodiments, the method comprises administering to the subject a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain 25 that targets CD5, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell wherein the endogenous CD5 gene has been knocked-out. In certain embodiments, the method comprises administering to the subject a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain that targets CD7, a transmembrane domain, and an intracellular domain, and administering to 30 the subject a second modified cell wherein the endogenous CD7 gene has been knocked-out. 2026223298   27 Aug 2026 In certain embodiments, the method comprises administering to the subject a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and administering to the subject a second modified cell wherein the endogenous CD5 gene has 5 been knocked-out. In certain embodiments, the method comprises administering to the subject a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and wherein the endogenous CD5 gene has been knocked-out in the cell. 10          In the various embodiments of the methods disclosed herein, the subject can be administered any of the CARs disclosed herein. The CAR can be specific for any tumor associated antigen (TAA) or tumor specific antigen (TSA) known to one of ordinary skill in the art. In certain embodiments, the CAR comprises a complementarity determining region 15 (CDR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36, 43-48, 53-58, 65-70, 83-88, and 95-100. In certain embodiments, the CAR comprises an antigen binding domain comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 41, 51, 63, 75, 81, and 93 and / or a light chain variable region comprising an amino acid sequence 20 selected from the group consisting of SEQ ID NOs: 30, 42, 52, 64, 76, 82, and 94. In certain embodiments, the CAR comprises an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 73, 74, 79, 80, 91, and 92. In certain embodiments, the subject is administered a CAR, wherein the CAR comprises a nucleic acid sequence encoded by any one of SEQ ID NOs: 1-13. In certain 25 embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 37, 38, 49, 59, 60, 71, 72, 77, 78, 89, and 90. In certain embodiments, the first and or second modified cell is a T cell. In certain embodiments, the cancer comprises a T cell lymphoma or a T cell leukemia. Types of cancers that can be treated with the compositions and methods of the present invention 30 include but are not limited to non-Hodgkins’s lymphoma and subtypes thereof including peripheral T-cell lymphoma (PTCL), Angioimmunoblastic T-cell lymphoma (AITL), 2026223298   27 Aug 2026 anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL, ALK-), Natural kil1er / T-cell lymphoma (NKTCL), Adult T-cell leukemia / lymphoma (ATLL), ALCL ALK+, enteropathy-type T-cell, hepatosplenic T-cell, subcutaneous panniculitisdike, and unclassifiable PTCL. 5          In certain embodiments, the endogenous gene (e.g. CD2, CDS, and CD7) is knocked- out using a CRISPR / Cas9 method. In certain embodiments, the CRISPR / Cas9 method utilizes an sgRNA targeting CD2, CD5, and / or CD7. In certain embodiments, the sgRNA comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 2224. 10          In certain embodiments, the CAR of 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 to a modified human FK-binding protein, allowing conditional dimerization using a smallmolecule drug (e.g. API903). When exposed to a synthetic dimerizing drug, the iCaspase9 15 becomes activated and leads to the rapid apoptosis of cells expressing this construct (e.g. the CAR T cell) (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 the CAR T cell, and upon expression it turns the non-toxic prodrug GCV into GCV-triphosphate, leading to cell death 20 by halting DNA replication. Compositions One aspect of the invention includes a composition comprising a first modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen 25 binding domain that targets CD2, a transmembrane domain, and an intracellular domain, and a second modified cell wherein the endogenous CD2 gene has been knocked-out. Another aspect of the invention includes a composition comprising a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain that targets CD5, a transmembrane domain, and an intracellular domain, and a second modified cell 30 wherein the endogenous CD5 gene has been knocked-out. 2026223298   27 Aug 2026 Yet another aspect of the invention includes a composition comprising a first modified cell comprising a CAR, wherein the CAR comprises an antigen binding domain that targets CD7, a transmembrane domain, and an intracellular domain, and a second modified cell wherein the endogenous CD7 gene has been knocked-out. 5          In certain embodiments, the CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-13. In certain embodiments, the endogenous genes have been knocked-out using a CRISPR / Cas system. In certain embodiments, the CRISPR / Cas9 system comprises a gRNA comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 22-24. 10          The invention further includes the composition of the invention and a pharmaceutically acceptable carrier. Chimeric Antigen Receptor (CAR) The present invention provides a chimeric antigen receptor (CAR) comprising an 15 antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the invention comprises a CAR comprising an antigen binding domain capable of binding CD2, a transmembrane domain, and an intracellular domain. Antigen binding domain 20          In one embodiment, the CAR of the invention comprises a target-specific binding element otherwise referred to as an antigen binding domain. The choice of antigen binding domain depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state (e.g. T cell 25 lymphoma or leukemia). In one embodiment, the CAR of the invention can be engineered to target a tumor antigen. The antigens discussed herein are merely included by way of example. The list is not intended to be exclusive and further examples will be readily apparent to those of skill in the art. Tumor antigens are proteins that are produced by tumor cells that elicit an immune 30 response, particularly T-cell mediated immune responses. The selection of the antigen binding domain of the invention will depend on the particular type of cancer to be treated. 2026223298   27 Aug 2026 Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RUI, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, 5 prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin. The type of tumor antigen referred to in the invention may also be a tumor-specific 10 antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may 15 be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells. Non-limiting examples of TSA or TAA antigens include the following: 20 Differentiation antigens such as MART-l / MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, 25 IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, 30 BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, 2026223298   27 Aug 2026 NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS. Depending on the desired antigen to be targeted, the CAR of the invention can be engineered to include the appropriate antigen binding domain that is specific to the desired 5 antigen target. For example, if CD2 is the desired antigen that is to be targeted, an antibody for CD2 can be used as the antigen binding domain for incorporation into the CAR of the invention. In certain embodiments, the antigen binding domain of the CAR targets CD2. In certain embodiments, the antigen binding domain of the CAR targets CD5. In certain 10   embodiments, the antigen binding domain of the CAR targets CD7. In some embodiments, the antigen binding domain in the CAR of the invention is anti-CD2 scFV. In some embodiments, the antigen binding domain in the CAR of the invention is anti-CD5 scFV. In some embodiments, the antigen binding domain in the CAR of the invention is anti-CD7 scFV. In some embodiments, the antigen binding domain is an 15 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. In certain embodiments, the antigen binding domain comprises a heavy chain variable region that comprises three heavy chain complementarity determining regions (HCDRs) and a light chain variable region that comprises three light chain complementarity determining 20 regions (LCDRs). In certain embodiments, the invention comprises a CAR comprising an antigen binding domain capable of binding CD2, wherein the antigen binding domain comprises a complementarity determining region (CDR) comprising the amino acid sequence of any one of SEQ ID NOs: 31,32, 33, 34, 35, 36, 43, 44, 45, 46, 47, 48, 53, 54, 55, 56, 57, 58, 65, 66, 25   67, 68, 69, or 70. In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD2, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 31, HCDR2 comprises the amino acid sequence of SEQ ID NO: 32, HCDR3 comprises the amino acid sequence of SEQ ID NO: 33, LCDR1 comprises the amino acid sequence of SEQ 30 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. 2026223298   27 Aug 2026 In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD2, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 43, HCDR2 comprises the amino acid sequence of SEQ ID NO: 44, HCDR3 comprises the amino acid sequence of SEQ ID NO: 45, LCDR1 comprises the amino acid sequence of SEQ 5 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. In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD2, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 65, HCDR2 comprises the amino acid sequence of SEQ ID NO: 66, HCDR3 comprises the 10 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. In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD2, wherein the antigen binding domain comprises a heavy chain variable region 15 comprising the amino acid sequence of SEQ ID NO: 29 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the antigen binding domain comprises a heavy 20 chain variable region comprising the amino acid sequence of SEQ ID NO: 51 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52. In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64. 25          In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD2, wherein the antigen binding domain is a scFv comprising the amino acid sequence set forth in any one of SEQ ID NOs: 27, 28, 39, 40, 50, 61, or 62. In certain embodiments, the invention comprises a CAR comprising an antigen binding domain capable of binding CD5, wherein the antigen binding domain comprises a 30 complementarity determining region (CDR) comprising the amino acid sequence of any one of SEQ ID NOs: 83, 84, 85, 86, 87, 88, 95, 96, 97, 98, 99, or 100. 2026223298   27 Aug 2026 In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD5, wherein 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 5 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. In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD5, wherein 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 10 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. In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD5, wherein the antigen binding domain comprises a heavy chain variable region 15 comprising the amino acid sequence of SEQ ID NO: 75 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 and / or a light chain variable region comprises the amino acid sequence of SEQ ID NO: 82. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID 20 NO: 93 and / or a light chain variable region comprises the amino acid sequence of SEQ ID NO: 94. In certain embodiments, the CAR comprises an antigen binding domain capable of binding CD5, wherein the antigen binding domain is a scFv comprising the amino acid sequence set forth in any one of SEQ ID NOs: 73, 74, 79, 80, 91, or 92. 25          Tolerable variations of the antigen binding domain sequences will be known to those of skill in the art. For example, in some embodiments the antigen binding domain comprises an amino acid sequence that has 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 30   96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 39, 40, 41, 42, 43, 2026223298   27 Aug 2026 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. Transmembrane domain 5          With respect to the transmembrane domain, the CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane 10 domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be 15 derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, 20 tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker. 25         In one embodiment, the transmembrane domain in the CAR of the invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 14. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 15. In another embodiment, the CD8 transmembrane domain comprises the amino 30 acid sequence of SEQ ID NO: 15. 2026223298   27 Aug 2026 In some instances, the transmembrane domain of the CAR of the invention comprises the CD8a hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 16. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 17. In another 5 embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 17. Between the antigen binding domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the 10 extracellular domain or, the cytoplasmic domain in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Intracellular domain 15          The intracellular domain or otherwise the cytoplasmic domain of the CAR of the invention is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term “intracellular 20 domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The 25 term intracellular domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal. Preferred examples of intracellular domains for use in the CAR of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any 30 derivative or variant of these sequences and any synthetic sequence that has the same functional capability. 2026223298   27 Aug 2026 It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary 5 cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as 10 immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of IT AM containing primary cytoplasmic signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma , CD3 delta , CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmic signaling molecule in the CAR of the invention 15 comprises a cytoplasmic signaling sequence derived from CD3 zeta. In a preferred embodiment, the intracellular domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired intracellular domain(s) useful in the context of the CAR of the invention. For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory 20 signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include, but are not limited to, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, 25 lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. Thus, while the invention is exemplified primarily with 4-1BB as the co-stimulatory signaling element, other costimulatory elements are within the scope of the invention. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the 30 CAR of the invention may be linked to each other in a random or specified order. 2026223298   27 Aug 2026 Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides a particularly suitable linker. In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the 5 intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB. In one embodiment, the intracellular domain in the CAR of the invention is designed to comprise the signaling domain of 4-IBB and the signaling domain of CD3-zeta, wherein 10 the signaling domain of 4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO: 18 and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 19. In one embodiment, the intracellular domain in the CAR of the invention is designed to comprise the signaling domain of 4-IBB and the signaling domain of CD3-zeta, wherein 15 the signaling domain of 4-IBB comprises a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 20 and the signaling domain of CD3-zeta comprises a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 21. In one embodiment, the intracellular domain in the CAR of the invention is designed to comprise the signaling domain of 4-IBB and the signaling domain of CD3-zeta, wherein 20 the signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 20 and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 21. In one embodiment, the anti-CD2 CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 26, 37, 38, 49, 59, or 60. In one embodiment, the anti-CD2 25 CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-7. In one embodiment, the anti-CD5 CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 71, 72, 77, 78, 89, or 90. In one embodiment, the anti-CD5 CAR is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8-13. 30          Tolerable variations of the CAR sequences will be known to those of skill in the art. For example, in some embodiments the CAR comprises an amino acid sequence that has at 2026223298   27 Aug 2026 least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences set forth in SEQ ID NO: 5   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 that has 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%, at least or 99% 10 sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 ,8, 9, 10, 11, 12, or 13. The invention should be construed to include any one of: a CAR, a nucleic acid encoding a CAR, a vector comprising a nucleic acid encoding a CAR, a cell comprising a CAR, a cell comprising a nucleic acid encoding a CAR, and a cell comprising a vector 15 comprising a nucleic acid encoding a CAR. CD2-MEDI507H2L-3028 CAR (SEQ ID NO: D ggatccCAAGTCCAACTGGTGCAATCAGGCGCAGAAGTCCAACGACCGGGGGCCAG TGTTAAAGTGTCTTGTAAAGCCTCCGGGTACATTTTTACTGAGTACTATATGTACT 20 GGGTCAGACAGGCCCCAGGGCAAGGTTTGGAACTTGTCGGACGCATAGATCCCG AAGACGGTTCTATAGATTACGTTGAGAAGTTCAAAAAGAAAGTCACACTTACTG CGGACACATCTAGTAGCACCGCATATATGGAACTGAGCAGTCTCACCTCAGACG ACACCGCAGTGTACTATTGCGCTCGCGGAAAGTTTAACTATAGGTTCGCGTACTG GGGACAGGGGACACTGGTGACTGTTAGCAGCggtggcggagggagcggcggtggaggaagcggag 25 gcggaggttccGACGTTGTGATGACGCAAAGTCCCCCGTCACTCCTTGTTACTCTCGGC CAGCCAGCGTCTATCTCTTGCCGGTCAAGCCAGAGCTTGCTCCACTCTAGTGGTA ACACGTATTTGAACTGGTTGCTGCAAAGGCCTGGACAATCTCCTCAGCCCCTGAT CTATTTGGTTAGCAAACTGGAAAGTGGTGTTCCAGACAGATTTTCAGGGTCTGGA TCAGGCACTGATTTCACTCTGAAGATCTCCGGGGTAGAGGCCGAGGACGTGGGA 30 GTCTATTACTGCATGCAGTTTACTCACTATCCTTATACCTTTGGTCAAGGGACGA AACTGGAGATCAAAtccgga CD2-OKT11H2L-3029 CAR (SEQ ID NO: 2) ggatccCAAGTTCAGCTTCAGCAACCAGGTGCTGAATTGGTCCGCCCTGGAACTAGC 3 5 GTTAAACTGTCTTGT AAGGC ATCCGGTT AT ACGTTT AC AAGTTATTGGATGC ACT GGATTAAGCAAAGGCCCGAACAAGGCCTTGAATGGATTGGGAGAATTGATCCCT ACGATAGCGAGACACACTACAATGAAAAATTTAAAGATAAGGCCATCCTCAGCG TAGATAAGAGCAGTTCTACCGCATACATACAGCTCTCAAGCCTGACGTCAGATG 2026223298   27 Aug 2026 ACTCAGCCGTTTATTATTGCTCAAGGCGGGACGCTAAATACGACGGCTATGCGCT TGACTACTGGGGACAAGGCACCACTTTGACAGTCTCCAGTggtggcggagggagcggcggt ggaggaagcggaggcggaggttccGATATAGTTATGACGCAAGCAGCACCCTCTGTACCTGT GACACCGGGTGAATCCGTTAGTATCTCATGCCGCTCTTCTAAAACCCTCTTGCAT 5 TCTAACGGCAATACATATTTGTATTGGTTCCTTCAACGACCAGGACAATCACCGC AAGTGCTTATTTATAGGATGTCTAACTTGGCTAGTGGGGTGCCAAATAGGTTCAG TGGGTCTGGATCTGAGACAACTTTCACGTTGAGAATAAGTAGGGTGGAAGCTGA AGACGTCGGTATATACTACTGTATGCAGCATTTGGAGTACCCTTACACTTTCGGG GGAGGTACTAAGCTCGAAATTAAAtccgga 10 CD2-OKT11L2H-3030 CAR (SEP ID NO: 3) ggatccGATATAGTTATGACGCAAGCAGCACCCTCTGTACCTGTGACACCGGGTGAA TCCGTTAGTATCTCATGCCGCTCTTCTAAAACCCTCTTGCATTCTAACGGCAATAC ATATTTGTATTGGTTCCTTCAACGACCAGGACAATCACCGCAAGTGCTTATTTAT 15 AGGATGTCTAACTTGGCTAGTGGGGTGCC AAATAGGTTCAGTGGGTCTGGATCTG AGACAACTTTCACGTTGAGAATAAGTAGGGTGGAAGCTGAAGACGTCGGTATAT ACTACTGTATGCAGCATTTGGAGTACCCTTACACTTTCGGGGGAGGTACTAAGCT CGAAATTAAAggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAAGTTCAGCTTCA GCAACCAGGTGCTGAATTGGTCCGCCCTGGAACTAGCGTTAAACTGTCTTGTAAG 20 GCATCCGGTTATACGTTTACAAGTTATTGGATGCACTGGATTAAGCAAAGGCCCG AACAAGGCCTTGAATGGATTGGGAGAATTGATCCCTACGATAGCGAGACACACT ACAATGAAAAATTTAAAGATAAGGCCATCCTCAGCGTAGATAAGAGCAGTTCTA CCGCATACATACAGCTCTCAAGCCTGACGTCAGATGACTCAGCCGTTTATTATTG CTCAAGGCGGGACGCTAAATACGACGGCTATGCGCTTGACTACTGGGGACAAGG 25 CACCACTTTGACAGTCTCCAGTtccgga CD2-T11-2-H2L-3031 CAR (SEP ID NO: 4) ggatccCAAGTTCAATTGCAGCAACCGGGTGCCGAGTTGGTAAGGCCCGGTGCGTC AGTCAAACTTAGTTGTAAAGCTAGTGGGTACACTTTTACTACGTTCTGGATGAAT 3 0 TGGGTGAAGC AACGACC AGGCC AAGGTCTGGAATGGATCGGC ATGATTGACCCG TCTGACTCAGAAGCTCATTACAACCAGATGTTCAAGGACAAGGCGACTCTGACT GTTGATAAAAGCTCAAGCACCGCCTACATGCAGCTCAGTAGCCTCACATCCGAG GATTCCGCAGTGTACTATTGCGCGAGGGGACGAGGGTATGATGACGGCGATGCG ATGGACTATTGGGGACAGGGGACCAGCGTAACAGTCAGTAGTggtggcggagggagcgg 35 cggtggaggaagcggaggcggaggttccGATATAGTTATGACCCAGTCTCCCGCCTCTCTGGCC GTTAGCTTGGGACAACGCGCTACCATCTCTTACCGAGCGTCTAAGTCCGTCAGTA CAAGCGGTTATAGTTACATGCACTGGAACCAGCAAAAGCCCGGACAACCTCCGA GACTCCTGATTTATTTGGTCTCTAACCTTGAGTCAGGTGTCCCAGCCAGATTCTCC GGCTCTGGAAGCGGCACTGACTTTACATTGAACATTCACCCCGTGGAGGAGGAA 40 GACGCTGCTACCTACTATTGCATGCAATTCACGCACTATCCCTACACATTCGGGG GGGGCACGAAATTGGAAATCAAAtccgga CD2-TS2-1811-H2L-3032 CAR (SEQ ID NO: 5) ggatccGAGGTTCAGCTTGAGGAGAGTGGGGGAGGTTTGGTAATGCCAGGTGGGTC 45 TTTGAAACTCAGTTGCGCGGCGTCAGGCTTCGCATTTTCCTCCTACGATATGTCCT GGGTCAGACAGACACCCGAGAAGCGGCTGGAATGGGTCGCTTACATTTCCGGGG 2026223298   27 Aug 2026 GAGGATTCACGTACTACCCGGATACAGTAAAGGGGAGATTTACTCTGAGCCGGG ACAACGCTAAGAATACCCTCTATCTCCAGATGTCCTCTTTGAAGAGTGAAGACAC AGCGATGTATTACTGTGCGAGACAAGGGGCCAATTGGGAGCTGGTTTACTGGGG CCAGGGGACGACATTGACGGTTTCTAGCggtggcggagggagcggcggtggaggaagcggaggcgg 5 aggttccGACATTGTAATGACACAATCACCTGCTACACTTAGCGTGACTCCAGGTGA TCGGGTATTCCTGAGCTGCCGCGCATCACAAAGTATATCCGACTTCCTGCACTGG TATCAGCAGAAATCTCACGAAAGTCCCAGGCTGCTGATTAAATACGCTTCCCAG AGTATTAGTGGTATCCCCTCACGATTTTCTGGCAGCGGGAGCGGTAGTGACTTCA CTCTTTCTATAAACTCCGTCGAGCCAGAAGACGTGGGGGTGTATCTTTGCCAAAA 10 TGGAC AC AATTTTCC ACC AACCTTTGGTGGGGGC ACC AAACTCGAAAT AAAGtccg ga CD2-TS2-1811-L2H-3033 CAR (SEP ID NO: 6) ggatccGACATTGTAATGACACAATCACCTGCTACACTTAGCGTGACTCCAGGTGAT 15 CGGGTATTCCTGAGCTGCCGCGC ATC ACAAAGTATATCCGACTTCCTGC ACTGGT ATCAGCAGAAATCTCACGAAAGTCCCAGGCTGCTGATTAAATACGCTTCCCAGA GTATTAGTGGTATCCCCTCACGATTTTCTGGCAGCGGGAGCGGTAGTGACTTCAC TCTTTCTATAAACTCCGTCGAGCCAGAAGACGTGGGGGTGTATCTTTGCCAAAAT GGACACAATTTTCCACCAACCTTTGGTGGGGGCACCAAACTCGAAATAAAGggtgg 20 cggagggagcggcggtggaggaagcggaggcggaggttccGAGGTT C AGC TT GAGG AGAGT GGGGGA GGTTTGGTAATGCCAGGTGGGTCTTTGAAACTCAGTTGCGCGGCGTCAGGCTTCG CATTTTCCTCCTACGATATGTCCTGGGTCAGACAGACACCCGAGAAGCGGCTGG AATGGGTCGCTTACATTTCCGGGGGAGGATTCACGTACTACCCGGATACAGTAA AGGGGAGATTTACTCTGAGCCGGGACAACGCTAAGAATACCCTCTATCTCCAGA 25 TGTCCTCTTTGAAGAGTGAAGACACAGCGATGTATTACTGTGCGAGACAAGGGG CCAATTGGGAGCTGGTTTACTGGGGCCAGGGGACGACATTGACGGTTTCTAGCtcc gga CD2-MEDI507L2H-3043 CAR (SEP ID NO: 7) 30 ggatccGACGTTGTGATGACGCAAAGTCCCCCGTCACTCCTTGTTACTCTCGGCCAG CCAGCGTCTATCTCTTGCCGGTCAAGCCAGAGCTTGCTCCACTCTAGTGGTAACA CGTATTTGAACTGGTTGCTGCAAAGGCCTGGACAATCTCCTCAGCCCCTGATCTA TTTGGTTAGCAAACTGGAAAGTGGTGTTCCAGACAGATTTTCAGGGTCTGGATCA GGCACTGATTTCACTCTGAAGATCTCCGGGGTAGAGGCCGAGGACGTGGGAGTC 3 5 T ATTACTGC ATGC AGTTT ACTC ACTATCCTT ATACCTTTGGTC AAGGGACGAAAC TGGAGATCAAAggtggcggagggagcggcggtggaggaagcggaggcggaggttccCAAGTCCAACTGG TGCAATCAGGCGCAGAAGTCCAACGACCGGGGGCCAGTGTTAAAGTGTCTTGTA AAGCCTCCGGGTACATTTTTACTGAGTACTATATGTACTGGGTCAGACAGGCCCC AGGGCAAGGTTTGGAACTTGTCGGACGCATAGATCCCGAAGACGGTTCTATAGA 40 TTACGTTGAGAAGTTCAAAAAGAAAGTCACACTTACTGCGGACACATCTAGTAG CACCGCATATATGGAACTGAGCAGTCTCACCTCAGACGACACCGCAGTGTACTA TTGCGCTCGCGGAAAGTTTAACTATAGGTTCGCGTACTGGGGACAGGGGACACT GGTGACTGTTAGCAGCtccgga 45   CD5-17L2H-3045 CAR (SEP ID NO: 8) 2026223298   27 Aug 2026 ggatccAACATTGTACTGACGCAAAGCCCCTCATCTTTGTCTGAGTCACTCGGCGGC AAAGTAACCATCACATGCAAGGCCAGTCAAGACATCAATAAATATATTGCTTGG TATCAGTATAAACCCGGCAAGGGGCCGCGACTGCTGATTCACTACACGAGTACC TTGCAACCGGGCATTCCGAGCCGATTTAGTGGCAGTGGCTCAGGTCGCGATTACT 5 CATTCTCAATAAGTAATCTCGAACCGGAAGACATAGCTACTTATTATTGCTTGCA GTACGATAATTTGTGGACCTTCGGGGGTGGTACAAAGTTGGAAATAAAGggtggcgg agggagcggcggtggaggaagcggaggcggaggttccGAGGT C C A AC T C GT AGA ATC AGGT C CC GG ATTGGTGCAACCATCCCAGAGCCTCTCTATTACATGCACGGTCTCTGGATTTAGT CTGACCAATTACGATGTGCATTGGGTGCGCCAGTCTCCCGGCAAGGGGTTGGAA 10 TGGCTTGGCGTTAT ATGGAACT ACGGAAAT AC AGACTAT AACGCCGCGTTT ATCT CTCGGCTGAGTATACGGAAAGACAGTAGTAAATCCCAGGTCTTTTTTACGATGTC ATCCCTGCAAACGCCAGATACCGCAATATATTACTGCGCCAGGAACCACGGTGA TGGTTATTATAATTGGTACTTCGATGTGTGGGGTACTGGCACTACAGTCACAGTA TCTTCAtctaga 15 CD5-9H2L-3048 CAR (SEP ID NO: 9) ggatcc CAG GTC CAG CTG AAA GAA AGC GGT CCA GAG CTG GAA AAA CCC GGT GCG AGC GTC AAA ATA TCA TGT AAA GCA AGC GGG TAT TCA TTC ACC GCG TAC TCT ATG AAC TGG GTT AAG CAA AAC AAC GGT ATG TCC TTG GAG 20 TGG ATA GGG TCT ATC GAC CCG TAT TAT GGG GAC ACA AAA TAC GCG CAG AAA TTC AAG GGG AAG GCC ACC CTG ACC GTA GAT AAA GCT AGT TCT ACT GCG TAC TTG CAA CTG AAA AGC CTC ACT TCT GAG GAC TCT GCC GTC TAC TAC TGT GCT CGG CGA ATG ATA ACG ACG GGG GAC TGG TAT TTC GAT GTT TGG GGT ACA GGG ACT ACG GTG ACT GTC AGT 25 AGCggtggcggagggagcggcggtggaggaagcggaggcggaggttcc CAT ATC GTC TTG ACT CAA TCA CCT AGT TCT TTG TCT GCG TCC CTT GGC GAC CGA GTC ACC ATA TCT TGC AGA GCG TCA CAG GAC ATT TCA ACG TAC CTC AAC TGG TAT CAG CAA AAA CCG GAC GGG ACT GTC AAG CTC TTG ATC TTC TAC ACT TCC AGA CTC CAC GCC GGG GTG CCA AGC AGA TTT AGT GGC TCT GGC AGC GGG ACA CAC 30 CAT AGT CTT ACA ATC AGC AAT CTT GAG CAA GAA GAC ATA GCC ACG TAT TTC TGC CAG CAA GGT AAC TCA CTT CCG TTC ACG TTT GGT AGT GGC ACC AAA CTG GAG ATA AAA tccgga CD5-9L2H-3049 CAR (SEP ID NO: 10) 3 5 Ggatcc CAT ATC GTC TTG ACT CAA TCA CCT AGT TCT TTG TCT GCG TCC CTT GGC GAC CGA GTC ACC ATA TCT TGC AGA GCG TCA CAG GAC ATT TCA ACG TAC CTC AAC TGG TAT CAG CAA AAA CCG GAC GGG ACT GTC AAG CTC TTG ATC TTC TAC ACT TCC AGA CTC CAC GCC GGG GTG CCA AGC AGA TTT AGT GGC TCT GGC AGC GGG ACA CAC CAT AGT CTT ACA ATC AGC AAT CTT GAG 40 CAA GAA GAC ATA GCC ACG TAT TTC TGC CAG CAA GGT AAC TCA CTT CCG TTC ACG TTT GGT AGT GGC ACC AAA CTG GAG ATA AAA ggtggcggagggagcggcggtggaggaagcggaggcggaggttcc CAG GTC CAG CTG AAA GAA AGC GGT CCA GAG CTG GAA AAA CCC GGT GCG AGC GTC AAA ATA TCA TGT AAA GCA AGC GGG TAT TCA TTC ACC GCG TAC TCT ATG AAC TGG GTT AAG CAA 45 AAC AAC GGT ATG TCC TTG GAG TGG ATA GGG TCT ATC GAC CCG TAT TAT GGG GAC ACA AAA TAC GCG CAG AAA TTC AAG GGG AAG GCC ACC CTG ACC 2026223298   27 Aug 2026 GTA GAT AAA GCT AGT TCT ACT GCG TAC TTG CAA CTG AAA AGC CTC ACT TCT GAG GAC TCT GCC GTC TAC TAC TGT GCT CGG CGA ATG ATA ACG ACG GGG GAC TGG TAT TTC GAT GTT TGG GGT ACA GGG ACT ACG GTG ACT GTC AGT AGC tccgga 5 CD5-34H2L-3052 CAR (SEP ID NO: 11) ggatccGAGGTTAAACTCGTGGAGAGCGGTGCCGAACTCGTCCGAAGTGGTGCTTC CGTTAAACTCAGTTGTGCCGCGTCAGGATTTAACATAAAAGATTACTACATTCAC TGGGTCAAACAGCGCCCGGAGCAGGGGCTTGAATGGATCGGGTGGATTGATCCT 10 GAAAACGGGCGC ACCGAAT ATGCTCCC AAGTTCC AGGGC AAAGCTACTATGACC GCTGACACCTCTAGTAACACTGCCTACCTGCAGTTGAGCTCTCTTACGTCTGAGG ATACCGCTGTGTACTACTGTAATAACGGAAATTATGTACGACACTATTACTTCGA CTACTGGGGGCAGGGCACTACTGTGACTGTATCTAGCggtggcggagggagcggcggtggagg aagcggaggcggaggttccGATTGGCTCACACAATCCCCTGCAATCCTGAGTGCATCTCCA 15 GGCGAGAAAGTAACTATGACTTGCAGAGCTATAAGCTCTGTGTCCTAC ATGC ACT GGTATCAGCAGAAGCCAGGTTCTTCCCCGAAGCCGTGGATATATGCTACAAGCA ATTTGGCATCCGGTGTTCCCGCCCGGTTTAGTGGCTCCGGTTCTGGGACAAGTTA CTCCCTCACGATCAGCAGGGTTGAAGCCGAGGACGCTGCCACTTACTATTGCCA ACAGTGGTCAAGTAACCCCAGGACTTTCGGGGGAGGAACTAAACTTGAAATCAA 20 Atctaga CD5-34L2H-3053 CAR (SEP ID NO: 12) Ggatcc GAT TGG CTC ACA CAA TCC CCT GCA ATC CTG AGT GCA TCT CCA GGC GAG AAA GTA ACT ATG ACT TGC AGA GCT ATA AGC TCT GTG TCC TAC ATG 25 CAC TGG TAT CAG CAG AAG CCA GGT TCT TCC CCG AAG CCG TGG ATA TAT GCT ACA AGC AAT TTG GCA TCC GGT GTT CCC GCC CGG TTT AGT GGC TCC GGT TCT GGG ACA AGT TAC TCC CTC ACG ATC AGC AGG GTT GAA GCC GAG GAC GCT GCC ACT TAC TAT TGC CAA CAG TGG TCA AGT AAC CCC AGG ACT TTC GGG GGA GGA ACT AAA CTT GAA ATC AAA 30 Ggtggcggagggagcggcggtggaggaagcggaggcggaggttcc GAG GTT AAA CTC GTG GAG AGC GGT GCC GAA CTC GTC CGA AGT GGT GCT TCC GTT AAA CTC AGT TGT GCC GCG TCA GGA TTT AAC ATA AAA GAT TAC TAC ATT CAC TGG GTC AAA CAG CGC CCG GAG CAG GGG CTT GAA TGG ATC GGG TGG ATT GAT CCT GAA AAC GGG CGC ACC GAA TAT GCT CCC AAG TTC CAG GGC AAA GCT ACT ATG ACC 35 GCT GAC ACC TCT AGT AAC ACT GCC TAC CTG CAG TTG AGC TCT CTT ACG TCT GAG GAT ACC GCT GTG TAC TAC TGT AAT AAC GGA AAT TAT GTA CGA CAC TAT TAC TTC GAC TAC TGG GGG CAG GGC ACT ACT GTG ACT GTA TCT AGC tCTAGA 40  CD5-17H2L-3054 CAR (SEP ID NO: 13) ggatccGAGGTCCAACTCGTAGAATCAGGTCCCGGATTGGTGCAACCATCCCAGAG CCTCTCTATTACATGCACGGTCTCTGGATTTAGTCTGACCAATTACGATGTGCATT GGGTGCGCCAGTCTCCCGGCAAGGGGTTGGAATGGCTTGGCGTTATATGGAACT ACGGAAATACAGACTATAACGCCGCGTTTATCTCTCGGCTGAGTATACGGAAAG 45 ACAGTAGTAAATCCCAGGTCTTTTTTACGATGTCATCCCTGCAAACGCCAGATAC CGCAATATATTACTGCGCCAGGAACCACGGTGATGGTTATTATAATTGGTACTTC 2026223298   27 Aug 2026 GATGTGTGGGGTACTGGCACTACAGTCACAGTATCTTCAggtggcggagggagcggcggtgg aggaagcggaggcggaggttccAACATTGTACTGACGCAAAGCCCCTCATCTTTGTCTGAGT CACTCGGCGGCAAAGTAACCATCACATGCAAGGCCAGTCAAGACATCAATAAAT ATATTGCTTGGTATCAGTATAAACCCGGCAAGGGGCCGCGACTGCTGATTCACTA 5 CACGAGTACCTTGCAACCGGGCATTCCGAGCCGATTTAGTGGCAGTGGCTCAGG TCGCGATTACTCATTCTCAATAAGTAATCTCGAACCGGAAGACATAGCTACTTAT TATTGCTTGCAGTACGATAATTTGTGGACCTTCGGGGGTGGTACAAAGTTGGAAA TAAAGtctaga 10 CD8 Transmembrane domain nucleic acid sequence (SEP ID NO: 14): atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact gc CD8 Transmembrane domain amino acid sequence (SEP ID NO: 15): 15 IYIWAPLAGTCGVLLLSLVITLYC CD8 hinge domain nucleic acid sequence (SEP ID NO: 16): accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 20 gacttcgcct gtgat CD8 hinge domain amino acid sequence (SEP ID NO: 17): TTTPAPRPPTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACD 25   4-1BB nucleic acid sequence (SEP ID NO: 18) aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt gaactg 30   CD3-zeta nucleic acid sequence (SEP ID ND: 19) agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 35 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgc 4-1BB amino acid sequence (SEP ID ND: 20): KRGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCEL 40 CD3-zeta amino acid sequence (SEP ID NO: 21): RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIG.MKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR 45 CD2-MEDI507H2L-3028 CAR amino acid sequence (SEP ID NO: 25) MALPVTALLLPLALLLHAARPGSPVPLVPSGAEVPRPGASVKVSCKASGYIFTEYY MYWVRP APGPGLELVGRIDPEDGSID YVEKFKKKVTLT ADTS S ST AYMELS SLTSD DTAVYYCARGKFNYRFAYWGPGTLVTVSSGGGGSGGGGSGGGGSDVVMTPSPPSL 50 LVTLGPPASISCRSSPSLLHSSGNTYLNWLLPRPGPSPPPLIYLVSKLESGVPDRFSGS 2026223298   27 Aug 2026 GSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKSGTTTPAPRPPTPAPT IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG RKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKF SRS AD AP AYKQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE 5 IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD2-MEDI507L2H-3043 CAR amino acid sequence (SEP ID NO: 26) MALPVTALLLPLALLLHAARPGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGN TYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYY 10 CMQFTHYPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVQRPGASVKV SCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSS T AYMELS SLTSDDT AVYYC ARGKFNYRF AYWGQGTLVT VS S SGTTTP APRPPTP APT IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG RKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVKF SRS AD AP AYKQGQN 15 QLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD2-MEDI507H2L-3028 scFv amino acid sequence (SEP ID NO: 27) GSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPE 20 DGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCARGKFNYRFAYWGQ GTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSG NTYLNWLLQRPGQSPQPLIYLVSKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVY YCMQFTHYPYTFGQGTKLEIKSG 25   CD2-MEDI507L2H-3043 scFv amino acid sequence (SEP ID NO: 28) GSDVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLV SKLESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIKG GGGSGGGGSGGGGSQVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAP GQGLELVGRIDPEDGSIDYVEKFKKKVTLTADTSSSTAYMELSSLTSDDTAVYYCAR 30 GKFNYRFAYWGQGTLVTVSSSG CD2-MEDI507 VH amino acid sequence (SEP ID NO: 29) QVQLVQSGAEVQRPGASVKVSCKASGYIFTEYYMYWVRQAPGQGLELVGRIDPED GSID YVEKFKKK VTLTADTS S STAYMELS SLTSDDT AVYYC ARGKFNYRF AYWGQG 35 TLVTVSS CD2-MEDI507 VL amino acid sequence (SEP ID NO: 30) DVVMTQSPPSLLVTLGQPASISCRSSQSLLHSSGNTYLNWLLQRPGQSPQPLIYLVSK LESGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQFTHYPYTFGQGTKLEIK 40 CD2-MEDI507 HCDR1 (SEP ID NO: 31) EYYMY CD2-MEDI507 HCDR2 (SEP ID NO: 32) 45 RIDPEDGSIDYVEKFKK 2026223298   27 Aug 2026 CD2-MEDI507 HCDR3 (SEP ID NO: 33) GKFNYRFAY CD2-MEDI507 LCDR1 (SEP ID NO: 34) 5 RSSPSLLHSSGNTYLN CD2-MEDI507 LCDR2 (SEP ID NO: 35) LVSKLES 10 CD2-MEDI507 LCDR3 (SEP ID NO: 36) MPFTHYPYT CD2-OKT11H2L-3029 CAR amino acid sequence (SEP ID NO: 37) MALPVTALLLPLALLLHAARPGSPVPLPPPGAELVRPGTSVKLSCKASGYTFTSYW 15 MHWIKpRPEpGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYipLSSLTSDDS AVYYC SRRDAKYDGYALDYWGPGTTLT VS SGGGGSGGGGSGGGGSDIVMTpAAP SVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLPRPGPSPPVLIYRMSNLASGVPNRF SGSGSETTFTLRISRVEAEDVGIYYCMPHLEYPYTFGGGTKLEIKSGTTTPAPRPPTPA PTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCK 20 RGRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKPG PNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEA YSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPR CD2-OKT11L2H-3030 CAR amino acid sequence (SEP ID NO: 38) 25 MALPVTALLLPLALLLHAARPGSDIVMTPAAPSVPVTPGESVSISCRSSKTLLHSNGN TYLYWFLPRPGPSPPVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYC MPHLEYPYTFGGGTKLEIKGGGGSGGGGSGGGGSPVPLPPPGAELVRPGTSVKLSC KASGYTFTSYWMHWIKPRPEPGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTA YipLS SLTSDD S AV YYC SRRDAKYDGYALDYWGPGTTLT VS S SGTTTP APRPPTP AP 3 0 TIASPPLSLRPEACRP AAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCKR GRKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKPGP NPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAY SEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPR 35 CD2-OKT11H2L-3029 scFv amino acid sequence (SEP ID NO: 39) GSPVPLPPPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKPRPEPGLEWIGRIDPY DSETHYNEKFKDKAILSVDKSSSTAYIPLSSLTSDDSAVYYCSRRDAKYDGYALDY WGPGTTLTVSSGGGGSGGGGSGGGGSDIVMTPAAPSVPVTPGESVSISCRSSKTLLH SNGNTYLYWFLPRPGPSPPVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDV 40 GIYYCMPHLEYPYTFGGGTKLEIKSG CD2-OKT11L2H-3030 scFv amino acid sequence (SEQ ID NO: 40) GSDIVMTPAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLPRPGPSPPVLIYRM SNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMPHLEYPYTFGGGTKLEIKGG 45 GGSGGGGSGGGGSPVPLPPPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKPRPE 2026223298   27 Aug 2026 QGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRD AKYDGYALD YWGPGTTLT VS S SG CD2-OKT11 VH amino acid sequence (SEP ID NO: 41) 5 QVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDS ETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWG QGTTLTVSS CD2-OKT11 VL amino acid sequence (SEP ID NO: 42) 10 DIVMTPAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLPRPGPSPPVLIYRMSN LASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMPHLEYPYTFGGGTKLEIK CD2-OKT11 HCDR1 (SEP ID NO: 43) SYWMH 15 CD2-OKT11 HCDR2 (SEP ID NO: 44) RIDPYDSETHYNEKFKD CD2-OKT11 HCDR3 (SEP ID NO: 45) 20 RDAKYDGYALDY CD2-OKT11 LCDR1 (SEP ID NO: 46) RS SKTLLHSNGNT YL Y 25 CD2-OKT11 LCDR2 (SEP ID NO: 47) RMSNLAS CD2-OKT11 LCDR3 (SEP ID NO: 48) MPHLEYPYT 30 CD2-T11-2-H2L-3031 CAR amino acid sequence (SEP ID NO: 49) MALPVTALLLPLALLLHAARPGSPVPLPPPGAELVRPGASVKLSCKASGYTFTTFW MNWVKPRPGPGLEWIGMIDPSDSEAHYNPMFKDKATLTVDKSSSTAYMPLSSLTS EDSAVYYCARGRGYDDGDAMDYWGPGTSVTVSSGGGGSGGGGSGGGGSDIVMTP 3 5 SP ASLAVSLGPRATIS YRASKS VSTSGYS YMHWNPPKPGPPPRLLIYLVSNLESGVP ARFSGSGSGTDFTLNIHPVEEEDAATYYCMPFTHYPYTFGGGTKLEIKSGTTTPAPRP PTPAPTIASPPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKPPFMRP VPTTPEEDGC SCRFPEEEEGGCELRVKF SRS AD AP AY KPGPNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKM 40 AEAYSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPR CD2-T11-2-H2L-3031 scFv amino acid sequence (SEP ID NO: 50) GSPVPLPPPGAELVRPGASVKLSCKASGYTFTTFWMNWVKPRPGPGLEWIGMIDP SDSEAHYNPMFKDKATLTVDKSSSTAYMPLSSLTSEDSAVYYCARGRGYDDGDAM 45 DYWGPGTSVTVSSGGGGSGGGGSGGGGSDIVMTPSPASLAVSLGPRATISYRASKS 2026223298   27 Aug 2026 VSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEE DAATYYCMQFTHYPYTFGGGTKLEIKSG CD2-T11-2-H2L-3031 VH amino acid sequence (SEP ID NO: 51) 5 QVQLQQPGAELVRPGASVKLSCKASGYTFTTFWMNWVKQRPGQGLEWIGMIDPSD SEAHYNQMFKDKATLTVDKS S STAYMQLS SLTSEDS AVYYCARGRGYDDGDAMD YWGQGTSVTVSS CD2-T11-2-H2L-3031 VL amino acid sequence (SEP ID NO: 52) 10 DIVMTPSPASLAVSLGPRATISYRASKSVSTSGYSYMHWNPPKPGPPPRLLIYLVSN LESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMPFTHYPYTFGGGTKLEIK CD2-T11-2 HCDRKSEQ ID NO: 53) TFWMN 15 CD2-T11-2 HCDR2 (SEP ID NO: 54) MIDPSDSEAHYNPMFKD CD2-T11-2 HCDR3 (SEP ID NO: 55) 20 GRGYDDGDAMDY CD2-T11-2 LCDR1 (SEP ID NO: 56) RASKSVSTSGYSYMH 25 CD2-T11-2 LCDR2 (SEP ID NO: 57) LVSNLES CD2-T11-2 LCDR3 (SEP ID NO: 58) MPFTHYPYT 30 CD2-TS2-18.1.1-H2L-3032 CAR amino acid sequence (SEP ID NO: 59) MALP VTALLLPLALLLHAARPGSEVPLEESGGGLVMPGGSLKLSC AASGF AF S SYD MSWVRPTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLPMSSLKSE DTAMYYCARPGANWELVYWGPGTTLTVSSGGGGSGGGGSGGGGSDIVMTPSPAT 35 LSVTPGDRVFLSCRASPSISDFLHWYPPKSHESPRLLIKYASPSISGIPSRFSGSGSGSD FTLSINSVEPEDVGVYLCPNGHNFPPTFGGGTKLEIKSGTTTPAPRPPTPAPTIASPPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKPPFMRP VPTTPEEDGC SCRFPEEEEGGCELRVKF SRS AD AP AYKPGPNPLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEIGMKGE 40 RRRGKGHDGLYPGLSTATKDTYDALHMPALPPR CD2-TS2-18.1.1-L2H-3033 CAR amino acid sequence (SEP ID NO: 60) MALPVTALLLPLALLLHAARPGSDIVMTPSPATLSVTPGDRVFLSCRASPSISDFLHW YPPKSHESPRLLIKYASPSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCPNGHNF 45 PPTFGGGTKLEIKGGGGSGGGGSGGGGSEVPLEESGGGLVMPGGSLKLSCAASGFA F S S YDMSWVRPTPEKRLEW VAYISGGGFT YYPDTVKGRFTLSRDNAKNTLYLPMS S 2026223298   27 Aug 2026 LKSEDTAMYYC ARQGANWELVYWGQGTTLTVS S SGTTTPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE 5 RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD2-TS2-18.E1-H2L-3032 scFv amino acid sequence (SEO ID NO: 61) GSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGG GFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWG 10 QGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVFLSCRASQSISDFL HWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGH NFPPTFGGGTKLEIKSG CD2-TS2-18.1.1-L2H-3033 scFv amino acid sequence (SEQ ID NO: 62) 15 GSDIVMTQ SPATES VTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISG IPSRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIKGGGGSGGG GSGGGGSEVQLEESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWV AYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWE L VYWGQGTTLT VS S SG 20 CD2-TS2-18.1.1 VH amino acid sequence (SEQ ID NO: 63) EVQLEESGGGLVMPGGSLKLSC AASGF AF S S YDMSWVRQTPEKRLEW VAYISGGGF TYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQG TTLTVSS 25 CD2-TS2-18.1.1 VL amino acid sequence (SEQ ID NO: 64) DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIP SRFSGSGSGSDFTLSINSVEPEDVGVYLCQNGHNFPPTFGGGTKLEIK 30   CD2-TS2-18.i l HCDR1 (SEQ ID NO: 65) SYDMS CD2-TS2-18.i l HCDR2 (SEQ ID NO: 66) YISGGGFTYYPDTVKG 35 CD2-TS2-18.i l HCDR3 (SEQ ID NO: 67) QGANWELVY CD2-TS2-18.i l LCDR1 (SEQ ID NO: 68) 40 RASQSISDFLH CD2-TS2-18.i l LCDR2 (SEQ ID NO: 69) YASQSIS 45   CD2-TS2-18.i l LCDR3 (SEQ IDNQ:70) QNGHNFPPT 2026223298   27 Aug 2026 CD5-17L2H-3045 CAR amino acid sequence (SEP ID NO:7D MALPVTALLLPLALLLHAARPGSNIVLTQSPSSLSESLGGKVTITCKASQDINKYIAW YQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNL WTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGPGLVQPSQSLSITCTVSGFSLT 5 NYDVHWVRQSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSQVFFTMSSLQ TPDTAIYYCARNHGDGYYNWYFDVWGTGTTVTVSSSRTTTPAPRPPTPAPTIASPPL SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYQQGQN PLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSE 10 IGMKGERRRGKGHDGLYpGLST ATKDTYD ALHMpALPPR CD5-17H2L-3054 CAR amino acid sequence (SEP ID NO:72) MALPVTALLLPLALLLHAARPGSEVPLVESGPGLVPPSPSLSITCTVSGFSLTNYDVH WVRPSPGKGLEWLGVIWNYGNTDYNAAFISRLSIRKDSSKSPVFFTMSSLPTPDTAI 15 YYCARNHGDGYYNWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSNIVLTPSPSSL SESLGGKVTITCKASPDINKYIAW YPYKPGKGPRLLIHYTSTLPPGIP SRF SGSGSGR DYSFSISNLEPEDIATYYCLPYDNLWTFGGGTKLEIKSRTTTPAPRPPTPAPTIASPPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGRKK LLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYPPGPNP 20 LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAYSEI GMKGERRRGKGHDGLYpGLSTATKDTYD ALHMpALPPR CD5-17L2H-3045 scFv amino acid sequence (SEP ID ND:73) NIVLTPSPSSLSESLGGKVTITCKASPDINKYIAWYPYKPGKGPRLLIHYTSTLPPGIP 25 SRFSGSGSGRDYSFSISNLEPEDIATYYCLPYDNLWTFGGGTKLEIKGGGGSGGGGS GGGGSEVPLVESGPGLVPPSPSLSITCTVSGFSLTNYDVHWVRPSPGKGLEWLGVI WNYGNTDYNAAFISRLSIRKDSSKSPVFFTMSSLPTPDTAIYYCARNHGDGYYNWY FD VWGTGTTVT VS S 30   CD5-17H2L-3054 scFv amino acid sequence (SEP ID NO:74) EVPLVESGPGLVPPSPSLSITCTVSGFSLTNYDVHWVRPSPGKGLEWLGVIWNYGN TDYNAAFISRLSIRKDSSKSPVFFTMSSLPTPDTAIYYCARNHGDGYYNWYFDVWG TGTTVTVSSGGGGSGGGGSGGGGSNIVLTPSPSSLSESLGGKVTITCKASPDINKYIA WYPYKPGKGPRLLIHYTSTLPPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLPYDN 35 LWTFGGGTKLEIK CD5-17 VH amino acid sequence (SEP ID ND:75) EVPLVESGPGLVPPSPSLSITCTVSGFSLTNYDVHWVRPSPGKGLEWLGVIWNYGN TDYNAAFISRLSIRKDSSKSPVFFTMSSLPTPDTAIYYCARNHGDGYYNWYFDVWG 40 TGTTVTVSS CD5-17 VL amino acid sequence (SEP ID ND:76) NIVLTPSPSSLSESLGGKVTITCKASPDINKYIAWYPYKPGKGPRLLIHYTSTLPPGIP SRFSGSGSGRDYSFSISNLEPEDIATYYCLPYDNLWTFGGGTKLEIK 45 CD5-9H2L-3048 CAR amino acid sequence (SEP ID NP:77) 2026223298   27 Aug 2026 MALPVTALLLPLALLLHAARPGSQVQLKESGPELEKPGASVKISCKASGYSFTAYSM NWVKQNNGMSLEWIGSIDPYYGDTKYAQKFKGKATLTVDKASSTAYLQLKSLTSE DSAVYYCARRMITTGDWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSHIVLTQSP SSLSASLGDRVTISCRASQDISTYLNWYQQKPDGTVKLLIFYTSRLHAGVPSRFSGSG 5 SGTHHSLTISNLEQEDIATYFCQQGNSLPFTFGSGTKLEIKSGTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK KLLYIFKQPFMRP VQTTQEEDGC SCRFPEEEEGGCELRVKF SRS AD AP AYKQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 10 CD5-9L2H-3049 CAR amino acid sequence (SEP ID NP:78) MALPVTALLLPLALLLHAARPGSHIVLTQSPSSLSASLGDRVTISCRASQDISTYLNW YQQKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEQEDIATYFCQQGNS LPFTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLKESGPELEKPGASVKISCKASGYSF 15 T AYSMNWVKQNNGMSLEWIGSIDP YYGDTKYAQKFKGKATLTVDKAS ST AYLQLK SLTSEDSAVYYCARRMITTGDWYFDVWGTGTTVTVSSSGTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK 20 GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD5-9H2L-3048 scFv amino acid sequence (SEP ID NO:79) GSPVPLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKPNNGMSLEWIGSIDPY YGDTKYAPKFKGKATLTVDKASSTAYLPLKSLTSEDSAVYYCARRMITTGDWYFD 25 VWGTGTTVTVSSGGGGSGGGGSGGGGSHIVLTPSPSSLSASLGDRVTISCRASPDIST YLNWYPPKPDGTVKLLIFYTSRLHAGVPSRFSGSGSGTHHSLTISNLEPEDIATYFCP PGNSLPFTFGSGTKLEIKSG CD5-9L2H-3049 scFv amino acid sequence (SEP ID ND:80) 30 HIVLTPSPSSLSASLGDRVTISCRASPDISTYLNWYPPKPDGTVKLLIFYTSRLHAGV PSRFSGSGSGTHHSLTISNLEPEDIATYFCPPGNSLPFTFGSGTKLEIKGGGGSGGGGS GGGGSPVPLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKPNNGMSLEWIGSI DPYYGDTKYAPKFKGKATLTVDKASSTAYLPLKSLTSEDSAVYYCARRMITTGDW YFD VWGTGTT VT VS S 35 CD5-9 VH amino acid sequence (SEP ID ND:81) PVPLKESGPELEKPGASVKISCKASGYSFTAYSMNWVKPNNGMSLEWIGSIDPYYG DTKYAPKFKGKATLTVDKASSTAYLPLKSLTSEDSAVYYCARRMITTGDWYFDVW GTGTTVTVSS 40 CD5-9 VL amino acid sequence (SEP ID ND:82) HIVLTPSPSSLSASLGDRVTISCRASPDISTYLNWYPPKPDGTVKLLIFYTSRLHAGV PSRFSGSGSGTHHSLTISNLEPEDIATYFCPPGNSLPFTFGSGTKLEIK 45  CD5-9 HCDR1 (SEP ID NP:83) AYSMN 2026223298   27 Aug 2026 CD5-9 HCDR2 (SEP ID NP:84) SIDPYYGDTKYAQKFKG 5  CD5-9 HCDR3 (SEP ID NP:85) RMITTGDWYFDV CD5-9 LCDR1 (SEP ID NP:86) RASPDISTYLN 10 CD5-9 LCDR2 (SEP ID NP:87) YTSRLHA CD5-9 LCDR3 (SEP ID NP:88) 15 PPGNSLPFT CD5-34H2L-3052 CAR amino acid sequence (SEP ID NP:89) MALPVTALLLPLALLLHAARPGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYI HWVKPRPEPGLEWIGWIDPENGRTEYAPKFPGKATMTADTSSNTAYLPLSSLTSED 20 TAVYYCNNGNYVRHYYFDYWGPGTTVTVSSGGGGSGGGGSGGGGSDWLTPSPAI LSASPGEKVTMTCRAISSVSYMHWYPPKPGSSPKPWIYATSNLASGVPARFSGSGSG TSYSLTISRVEAEDAATYYCPPWSSNPRTFGGGTKLEIKSRTTTPAPRPPTPAPTIASP PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRGR KKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYPPGP 25 NPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEAY SEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPR CD5-34L2H-3053 CAR amino acid sequence (SEP ID ND: 90) MALPVTALLLPLALLLHAARPGSDWLTPSPAILSASPGEKVTMTCRAISSVSYMHW 30 YPPKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCPPWS SNPRTFGGGTKLEIKGGGGSGGGGSGGGGSEVKLVESGAELVRSGASVKLSCAASG FNIKDYYIHWVKPRPEPGLEWIGWIDPENGRTEYAPKFPGKATMTADTSSNTAYLP LS SLT SEDT AVYYCNNGNYVRHYYFD YWGPGTT VT VS S SRTTTP APRPPTP APTIAS PPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHMKRG 35 RKKLLYIFKPPFMRPVPTTPEEDGCSCRFPEEEEGGCELTSRVKFSRSADAPAYPPG PNPLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPPEGLYNELPKDKMAEA YSEIGMKGERRRGKGHDGLYPGLSTATKDTYDALHMPALPPR CD5-34H2L-3052 scFv amino acid sequence (SEP ID ND: 91) 40 EVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKPRPEPGLEWIGWIDPENG RTEYAPKFPGKATMTADTSSNTAYLPLSSLTSEDTAVYYCNNGNYVRHYYFDYWG PGTTVTVSSGGGGSGGGGSGGGGSDWLTPSPAILSASPGEKVTMTCRAISSVSYMH WYPPKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCPP WSSNPRTFGGGTKLEIK 45 CD5-34L2H-3053 scFv amino acid sequence (SEP ID NO: 92) 2026223298   27 Aug 2026 DWLTQSPAILSASPGEKVTMTCRAISSVSYMHWYQQKPGSSPKPWIYATSNLASGVP ARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPRTFGGGTKLEIKGGGGSGGGG SGGGGSEVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGW IDPENGRTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYY 5 FDYWGQGTTVTVSS CD5-34 VH amino acid sequence (SEP ID NO: 93) EVKLVESGAELVRSGASVKLSCAASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENG RTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNNGNYVRHYYFDYWG 10 QGTTVTVSS CD5-34 VL amino acid sequence (SEP ID NO: 94) DWLTPSPAILSASPGEKVTMTCRAISSVSYMHWYPPKPGSSPKPWIYATSNLASGVP ARFSGSGSGTSYSLTISRVEAEDAATYYCPPWSSNPRTFGGGTKLEIKSR 15 CD5-34 HCDR1 (SEP ID NO: 95) DYYIH CD5-34 HCDR2 (SEP ID NO: 96) 20 WIDPENGRTEYAPKFPG CD5-34 HCDR3 (SEP ID NO: 97) GNYVRHYYFDY 25   CD5-34 LCDR1 (SEP ID NO: 98) RAISSVSYMH CD5-34 LCDR2 (SEP ID NO: 99) ATSNLAS 30 CD5-34 LCDR3 (SEP ID NO: 100) PPWSSNPRT 35 CRISPR / Cas Certain embodiments of the invention include cells that have been modified by a CRISPR / Cas system. CRISPR / Cas systems include, but are not limited to, the CRISPR / Cas9 system and the CRISPR / Cpf1 system. In certain embodiments, the invention includes cells that have been modified using the CRISPR / Cas9 system. In certain embodiments, the 40 modifications include knocking-out or mutating an endogenous gene, e.g. CD2, CD5, or CD7. 2026223298   27 Aug 2026 The CRISPR / Cas9 system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a ‘seed’ sequence within the guide RNA (gRNA or sgRNA) and a conserved tri-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR / Cas9 5 system can thereby be engineered 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. The CRISPR / Cas system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes. 10          One example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Publication No. US2014 / 0068797, which is incorporated herein by reference in its entirety. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks 15 endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes. CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for 20 a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. In certain embodiments, the CRISPR system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not 25 limited to Cpfl, T7, Cas3, Cas8a, Cas8b, CaslOd, Csel, Csyl, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combination thereof. In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter, such as one that is 30 inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline). However, it should be appreciated that other inducible promoters can 2026223298   27 Aug 2026 be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector. The guide nucleic acid sequence is specific for a gene and targets that gene for Cas 5 endonuclease-induced double strand breaks. The sequence of the guide nucleic acid sequence may be within a loci of the gene. In one embodiment, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, 25,26, 27,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length. The guide nucleic acid sequence may be specific for any gene, e.g. CD2, CD5, CD7. 10 The guide nucleic acid sequence includes a RNA sequence, a DNA sequence, a combination thereof (a RNA-DNA combination sequence), or a sequence with synthetic nucleotides. The guide nucleic acid sequence can be a single molecule or a double molecule. In one embodiment, the guide nucleic acid sequence comprises a single guide RNA. In the context of formation of a CRISPR complex, “target sequence” refers to a 15 sequence to which a guide sequence is designed to have some complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA 20 polynucleotides. In certain embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or nucleus. Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in 25 cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As with the target sequence, it is believed that complete complementarity is not needed, provided this is sufficient to be functional. In certain embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when 30 optimally aligned. 2026223298   27 Aug 2026 In other embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a 5 tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable 10 orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate 15 sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In certain embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 20   9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase 25 activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In certain embodiments, a tagged CRISPR enzyme is used to identify the location of a target 30 sequence. 2026223298   27 Aug 2026 Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a 5 transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Another delivery mode for the CRISPR / Cas9 comprises 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 and RNA viruses, which have either episomal or integrated 10 genomes after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu et al., 1994, Gene Therapy 1:13-26). In certain embodiments, the CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other 15 species. In certain embodiments, Cas9 can include: spCas9, Cpfl, CasY, CasX, or saCas9. In general, CRISPR / Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with the guiding RNA. CRISPR / Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein 20 interaction domains, dimerization domains, as well as other domains. The CRISPR / Cas proteins can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. In certain embodiments, the CRISPR / Cas-like protein of the fusion protein can be derived from a wild type Cas9 protein or fragment thereof. In other embodiments, the CRISPR / Cas can be derived from 25 modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, and so forth) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein. In general, a Cas9 protein comprises at least two nuclease 30   (i.e., DNase) domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC and HNH domains work together to 2026223298   27 Aug 2026 cut single strands to make a double-stranded break in DNA. (Jinek et al., 2012, Science, 337:816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain). For example, the Cas9-derived protein can be modified such that one of the nuclease 5 domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent). In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein is able to introduce a nick into a double-stranded nucleic acid (such protein is termed a “nickase”), but not cleave the double-stranded DNA. In any of the above-described embodiments, any or all of the nuclease domains can be inactivated by one or more deletion 10 mutations, insertion mutations, and / or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art. In one non-limiting embodiment, a vector drives the expression of the CRISPR system. The art is replete with suitable vectors that are useful in the present invention. The 15 vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The vectors of the present invention may also be used for nucleic acid standard gene delivery protocols. Methods for gene delivery are known in the art (U.S. Patent Nos. 5,399,346, 20   5,580,859 & 5,589,466, incorporated by reference herein in their entireties). Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4th Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012), and in other virology and molecular biology manuals. Viruses, which are 25 useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193). 30 2026223298   27 Aug 2026 Introduction of Nucleic Acids Methods of introducing nucleic acids into a cell include physical, biological and chemical methods. Physical methods for introducing a polynucleotide, such as RNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, 5 microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). RNA can also be introduced into cells using cationic liposome mediated 10 transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001). Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, 15 have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362. Chemical means for introducing a polynucleotide into a host cell include colloidal 20 dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Lipids suitable for use can be obtained from commercial sources. For example, 25 dimyristyl phosphatidylcholine (“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; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can 30 be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single 2026223298   27 Aug 2026 and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when 5 phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as 10 nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes. Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed. Such 15 assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELIS As and Western blots) or by assays described herein to identify agents falling within the scope of the invention. 20          Moreover, the nucleic acids may be introduced by any means, such as transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells. One nucleic acid may be introduced by one method and another nucleic acid may be introduced into the T cell by a different method. RNA 25          In one embodiment, the nucleic acids introduced into the T cell are RNA. In another embodiment, the RNA is mRNA that comprises in vitro transcribed RNA or synthetic RNA. The 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. 30 The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. The desired 2026223298   27 Aug 2026 template for in vitro transcription is a chimeric membrane protein. By way of example, the template encodes an antibody, a fragment of an antibody or a portion of an antibody. By way of another example, the template comprises an extracellular domain comprising a single chain variable domain of an antibody, such as anti-CD3, and an intracellular domain of a co-5 stimulatory molecule. In one embodiment, the template for the RNA chimeric membrane protein encodes a chimeric membrane protein comprising an extracellular domain comprising an antigen binding domain derived from an antibody to a co-stimulatory molecule, and an intracellular domain derived from a portion of an intracellular domain of CD28 and 4-IBB. PCR can be used to generate a template for in vitro transcription of mRNA which is 10 then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. “Substantially complementary”, as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. 15 Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a gene that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs. The primers can also be designed to amplify 20 a portion of a gene that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs. Primers useful for PCR are generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are 25 upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3' to the 30 DNA sequence to be amplified relative to the coding strand. 2026223298   27 Aug 2026 Chemical structures that have the ability to promote stability and / or translation efficiency of the RNA may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, 5 including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA. The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the 10 gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR 15 sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art. In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being 20 added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments the 5' UTR can be derived from an RNA virus whose RNA genome is 25 stable in cells. In other embodiments various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA. To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is 30 added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be 2026223298   27 Aug 2026 transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art. 5          In one embodiment, the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized 10 mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription. On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003). 15          The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a 20 method which allows construction of DNA templates with polyA / T 3' stretch without cloning highly desirable. The polyA / T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro 25 recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines. Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli poly A polymerase (E-PAP). In one 30 embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. 2026223298   27 Aug 2026 Additionally, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain 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 the stability of the RNA. 5          5' caps also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)). 10          The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, 15 lipids, proteins, antioxidants, and surfactants can be included. In some embodiments, the RNA is electroporated into the cells, such as in vitro transcribed RNA. The disclosed methods can be applied to the modulation of T cell activity in basic research and therapy, in the fields of cancer, stem cells, acute and chronic infections, and 20 autoimmune diseases, including the assessment of the ability of the genetically modified T cell to kill a target cancer cell. The methods also provide the ability to control the level of expression over a wide range by changing, for example, the promoter or the amount of input RNA, making it possible to individually regulate the expression level. Furthermore, the PCR-based technique 25 of mRNA production greatly facilitates the design of the mRNAs with different structures and combination of their domains. One advantage of RNA transfection methods of the invention is that RNA transfection is essentially transient and a vector-free. A RNA transgene can be delivered to a lymphocyte and expressed therein following a brief in vitro cell activation, as a minimal 30 expressing cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely. Cloning of 2026223298   27 Aug 2026 cells is not necessary because of the efficiency of transfection of the RNA and its ability to uniformly modify the entire lymphocyte population. Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA) makes use of two different strategies both of which have been successively tested in various animal 5 models. Cells are transfected with in vitro-transcribed RNA by means of lipofection or electroporation. It is desirable to stabilize IVT-RNA using various modifications in order to achieve prolonged expression of transferred IVT-RNA. Some IVT vectors are known in the literature which are utilized in a standardized manner as template for in vitro transcription and which have been genetically modified in 10 such a way that stabilized RNA transcripts are produced. Currently protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest which is flanked either 3' and / or 5' by untranslated regions (UTR), and a 3' polyadenyl cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of 15 the polyadenyl cassette by type II restriction enzymes (recognition sequence corresponds to cleavage site). The polyadenyl cassette thus corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and extend or mask the poly(A) sequence at the 3' end. It is not clear, whether this nonphysiological overhang affects the amount of protein produced 20 intracellularly from such a construct. RNA has several advantages over more traditional plasmid or viral approaches. Gene expression from an RNA source does not require transcription and the protein product is produced rapidly after the transfection. Further, since the RNA has to only gain access to the cytoplasm, rather than the nucleus, and therefore typical transfection methods result in an 25 extremely high rate of transfection. In addition, plasmid based approaches require that the promoter driving the expression of the gene of interest be active in the cells under study. In another aspect, the RNA construct is delivered into the cells by electroporation. See, e.g., the formulations and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 30   2005 / 0070841 Al, US 2004 / 0059285A1, US 2004 / 0092907A1. The various parameters including electric field strength required for electroporation of any known cell type are 2026223298   27 Aug 2026 generally known in the relevant research literature as well as numerous patents and applications in the field. See e.g., U.S. Pat. No. 6,678,556, U.S. Pat. No. 7,171,264, and U.S. Pat. No. 7,173,116. Apparatus for therapeutic application of electroporation are available commercially, e.g., the MedPulser™ DNA Electroporation Therapy System 5 (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. No. 6,233,482; electroporation may also be used for transfection of cells in vitro as described e.g. in US20070128708A1. Electroporation may also be utilized to deliver nucleic acids into cells in vitro. Accordingly, electroporation- 10 mediated administration into cells of nucleic acids including expression constructs utilizing any of the many available devices and electroporation systems known to those of skill in the art presents an exciting new means for delivering an RNA of interest to a target cell. Sources of T Cells 15          In certain embodiments, a source of T cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain embodiments, any number of T cell lines 20 available in the art, may be used. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated 25 white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, 30 for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. 2026223298   27 Aug 2026 In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative 5 selection techniques. The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19 and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell 10 bound antibody. Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface 15 markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CDllb, CD16, HLA-DR, and CD8. For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain 20 embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a 25 concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. 30          T cells can also be frozen after the washing step, which does not require the monocyte-removal step. While not wishing to be bound by theory, the freeze and subsequent 2026223298   27 Aug 2026 thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, 5 one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen. 10          In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells. 15 Expansion of T Cells In certain embodiments, the T cells disclosed herein can be multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 20   3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold. Following culturing, the T cells can be incubated in cell medium in a culture 25 apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or greater before passing the cells to another culture apparatus. More preferably, the level of confluence is 90% or greater. A period of 30 time can be any time suitable for the culture of cells in vitro. The T cell medium may be replaced during the culture of the T cells at any time. Preferably, the T cell medium is 2026223298   27 Aug 2026 replaced about every 2 to 3 days. The T cells are then harvested from the culture apparatus whereupon the T cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing nucleic acids into the T cell. 5          In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with the RNA encoding the chimeric membrane protein. Another procedure for ex vivo expansion cells is described in U.S. Pat. No. 5,199,942 10 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No. 5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of T cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding the T cells comprises culturing the T cells 15 with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand. The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 20    1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days. Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium 25 under conditions that facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time. Each round of subculturing is referred to as a passage. When cells are subcultured, 30 they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For 2026223298   27 Aug 2026 example, a cultured cell population that has been passaged ten times may be referred to as a PIO culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary culture (P2 5 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time 10 between passaging. In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, 15 including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a. or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, 20 MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be 25 infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2). The medium used to culture the T cells may include an agent that can co-stimulate the T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent 30 that can stimulate CD28 is an antibody to CD28. This is because, as demonstrated by the data disclosed herein, a cell isolated by the methods disclosed herein can be expanded 2026223298   27 Aug 2026 approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In one embodiment, 5 the T cells expand in the range of about 20 fold to about 50 fold, or more by culturing the electroporated population. In one embodiment, the method of expanding the T cells can further comprise isolating the expanded T cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded T cells 10 followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, into the expanded population of T cells, wherein the agent further stimulates the T cell. The agent may stimulate the T cells, such as by stimulating further expansion, effector function, or another T 15 cell function. Pharmaceutical compositions Pharmaceutical compositions of the present invention may comprise the modified cell or population of cells as described herein, in combination with one or more pharmaceutically 20 or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the 25 present invention are preferably formulated for intravenous administration. Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by 30 clinical trials. 2026223298   27 Aug 2026 The cells of the invention to be administered may be autologous, allogeneic or xenogeneic with respect to the subject undergoing therapy. Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell 5 compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art. It can generally be stated that a pharmaceutical composition comprising the modified T cells described herein may be administered at a dosage of 104 to 109 cells / kg body weight, 10 in some instances 105 to 106 cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by 15 one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. The administration of the modified cells of the invention may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, 20 implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like. 25          It should be understood that the method and compositions that would be useful in the present invention are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, and therapeutic methods of the invention, and are not intended to limit the scope of 30 what the inventors regard as their invention. 2026223298   27 Aug 2026 The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, 5 “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications 10 and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. 15 EXPERIMENTAL EXAMPLES The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the 20 teachings provided herein. The materials and methods employed in these experiments are now described. Target gRNA ID DNA seq Position Strand Sequence PAM On CD2 #8 Ex3 116760612 - ACAGCTGACAGGCTCGACAC (SEQ ID NO: 22) TGG 63.4 CD5 #4 Ex2- In2 61115090 + CGGCTCAGCTGGTATGACCC (SEQ ID NO: 23) AGG 66.7 CD7 #85 Ex2 670 - GGAGCAGGTGATGTTGACGG (SEQ ID NO: 24) AGG 74.9 sgRNAs were designed to target CD2, CD5, or CD7 (e.g. SEQ ID NOs: 22-24, respectively) and synthesized using the GeneArt Precision sgRNA synthesis kit. Cas9 expression plasmid (pGEM-Cas9) was amplified and linearized. Cas9 RNA was synthesized 25 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. Expression of CD2 / CD5 / CD7 on Jurkat cells was detected by flow cytometry and the most 2026223298   27 Aug 2026 effective CD2 / CD5 / CD7 sgRNAs were determined. CRISPR editing was then performed in primary human T cells using the most efficient CD2 / CD5 / CD7 sgRNA: the chosen sgRNA and Cas9 RNA were electroporated into primary human T cells. CD2 / CD5 / CD7 expression was detected on the primary human T cells by flow cytometry to validate the knock- 5 out / editing efficiency. Specifically, fresh CD4 / CD8 T cells were obtained and incubated with dynabeads on day 0. On day 4, cells were de-beaded then electroporated with Cas9 and sgRNA. Conditioned media (TCM (X-vivol5, human serum 5%, Glutamine), IL-7 lOng / ml, and IL-15 lOng / ml) was added to the cells. On day 6, cells were transduced with CAR lentivirus. On 10 day 9, CAR expression was assessed. Cells were fed to 0.8e6 / ml and frozen when volume <300 fl (FIG. 22). CAR constructs: All constructs were generated using the lentiviral pTRPE 4-1BB CD3zeta backbone. OKT11 CARs and TS2 / 18.1.1 CARs were constructed using scFvs from antibodies generated from hybridomas purchased from ATCC (ATCC® CRL-8027™ and 15 ATCC® HB-195™, respectively). The T11-2 CAR was constructed using an scFv from an antibody generated from a hybridoma that was received from Ellis Reinherz. All CD5 CARs were constructed using scFvs from antibody sequences published in WO 2010 / 022737 Al, contents of which are incorporated by reference in their entirety herein. 20          The results of the experiments are now described. Example 1: A novel approach to target T cell lymphomas and leukemias without causing T cell toxicity T-cell lymphomas and leukemias have an overall very poor prognosis, and there are few therapeutic options available for these patients. Chimeric antigen receptor T cell (CART) 25 immunotherapy has led to unprecedented results in CD 19+ B-cell non-Hodgkin lymphoma (B-NHL). Herein, another successful “CART19-like” product was designed to target T-NHL. As CD19 is not expressed in T-NHL, additional targets like CD2, CD5, CD7 and others were evaluated for CART therapy. However, all these targets are also expressed by normal T cells, leading to unacceptable clinical toxicity (T cell aplasia - immunodeficiency) (FIG. 1). 30 Herein, a safe and effective CART strategy was developed for the treatment of T-cell lymphomas by editing the normal T cells to be resistant to CART killing (FIG. 2). CART 2026223298   27 Aug 2026 therapy against T-cell lymphomas and leukemias was feasible when the CART target (CD2, CD5, CD7) was temporarily removed from normal T cells, thus avoiding CART-mediated killing and immunodeficiency (FIG. 2). 5 Example 2: Anti-CD5 CAR T cells (CART5) and CD5 knocked-out (KO) normal T cells A two-pronged immunotherapy is disclosed herein that includes anti-CD5 CAR T cells (CART5) and CD5 knocked-out (KO) normal T cells (FIG. 3). The CART5 destroys T cell lymphoma (e.g. T-NHL) or T cell leukemia cells but also kills normal T cells. The infusion of CD5 KO normal T cells provides CART-resistant T cell immunity until CART5 10 cells are depleted, in some cases by using a suicide gene (e.g. iCasp9, CD20 / rituximab or others). CD5 was selected as a T-NHL target due to high expression on T-NHL cells and absent expression in other tissues besides T cells and a minor B cell subset. Six anti-CD5 CAR constructs were generated using single-chain variable fragments (scFv) with different 15 affinity (#17, #34, #9 with high, medium, low affinity respectively (Klitgaard JL, et al. (2013) British journal of haematology 163:182-93)) and expressed in T cells (FIGs. 4-5). Intriguingly, anti-CD5 CART did not require the knockout of CD5 to manufacture CART5 cells despite CD5 being expressed in 100% of CART cells (Mamonkin M, et. al. (2015) Blood 126:983-92), although CD5 expression was lower as compared to control T cells (FIG. 20   8). Without CRISPR-Cas9 KO of CD5, the CD5 mean fluorescence intensity (MFI) was 10 fold less in CART5 as compared to control T cells, while there was no change in another pan T-cell marker such as CD2 (FIG. 8). In vitro and in vivo activity of the different CART5 constructs were compared. Construct C3054, which was derived from the high-affinity scFv #17, demonstrated the best 25 in vivo killing. Jurkat cells were transduced with different CAR5 constructs (FIG. 13, targeted epitope and affinity shown to the left) and with a GFP-NFAT reporter then cocultured with CD5+ tumor cells (or controls) for 24 hours. The lead CART5 (C3054) shows increased NF AT activation (FIG. 13). The lead anti-CD5 CART was implemented using a suicide system, and its function 30 was tested in vitro and in vivo. Without wishing to be bound by specific theory, removal of 2026223298   27 Aug 2026 CD5 (CRISPR-Cas9 KO) further increases CART5 anti-turnor effect by eliminating the possible in cis surface interaction between CAR5 and CD5 on the CART5. Insertion of a suicide system in the lead CART5 product: The lead candidate CART5 (C3054) products are engineered to express a suicide system (FIG. 26). A P2A bicistronic 5 vector encoding for both the CAR5 and the inducible-caspase9 suicide system (Di Stasi A, et al. (2011) 365:1673-83) (iCART5) is developed. Both orientations are cloned, CAR5-P2A-iCasp9 and iCasp9-P2A-CAR5 (FIG. 26), to define the most efficient one (higher % doubleexpressing cells and lower %CAR5+ iCasp9- for safety). iCART5 is efficiently eliminated using the clinical-grade compound rimiducid (AP1903, Bellicum Pharmaceuticals). 10         In vitro testing of iCART5: The newly generated iCART5 is compared to WT CART5 to confirm their efficacy (in vitro luciferase-based killing) and phenotype / function upon antigen stimulation (CD5+ Jurkat T-leukemia cells) (flow cytometry phenotype, 30-plex cytokine analysis by Luminex assay, CFSE proliferation and CD 107a degranulation). Importantly, in vitro depletion is tested by co-culturing iCART5 with different concentrations 15   of rimiducid (0, 0.03, 0.3, 3, 10 nM) and checking killing at 15’, 30’ and at 2, 6, 12 and 24 hours. iCART5 is also tested against primary T-NHL cells using an established killing assay using primary CFSE-labelled Sezary cells. In vivo testing of iCART5: In vivo xenograft models (Ruella M, et al. (2016) J Clin Invest) [using the click-beetle green (CBG)+ T-leukemia cell line Jurkat cell line and click-20 beetle red (CBR)+ iCART5] are used to test the ability of rimiducid (50ug / mouse 23) to deplete iCART5 vs. WT CART5 in vivo. NOD SCID gamma-deficient mice (NSG) mice (8 per group) are injected with 2xl0e6 CART5 cells / mouse. Tumor burden over time is assessed as bioluminescence (CBG) and T cell phenotype is studied by flow cytometry and expansion by bioluminescence (CBR) at multiple time points (hours / days). Mice are kept long-term (3 25   4 months) for survival and monitoring of relapse. A human T-NHL xenograft model was previously establised by injecting primary Sezary cells i.v.. This model is used to test the iCART5 for both anti-tumor and depletion efficacies. Evaluation of the role of CD5 KO in CART5: Preliminary data demonstrated that CD5 CART5 are more effective than WT CART5 in vivo. CD5 is knocked out on CART5 30 cells using CRISPR-Cas9. The CRISPR-Cas9 CART expansion protocol was previously optimized. Wild-type CART5 is compared to CD5 KO CART5 in vitro by testing CART5 2026223298   27 Aug 2026 viability, antigen-driven proliferation (using CFSE labeling), cytokine production (by 30-plex Luminex), degranulation (CD 107a assessment by flow cytometry), cytotoxicity (luciferase-based), and phenotype (memory subsets, Thl / Th2)., Both cell lines (e.g., Jurkat) and primary samples are used as targets. In vivo comparison of CD5 KO vs. WT CART5 5 (lxl0e6 cells / mouse) is performed in NSG mice bearing Jurkat and monitoring expansion and phenotype in the peripheral blood at day 10 and 14. Mechanism of enhancement of CART5 function by CD5 KO: Having proven that CD5KO improves CART5 activity, additional studies are performed to understand the mechanism: i. confocal imaging to analyze the localization of CAR5 and CD5 on CART5 10 cells; ii. single-molecule imaging (ONI nanoimager) to prove that CAR5 binds in cis to CD5; iii. expressing CAR5 in CD5+Jurkat and show that CART5 fail to kill CAR5+ Jurkat because the CD5 epitope is masked (by CAR5); and iv. as CD5 as an inhibitory role on T cell activation, we will study CART5 activation in the presence or not of CD5 (phospo-flow cytometry). 15          Generating CART-resistant normal T cells to avoid T cell aplasia: CART5 are unable to distinguish neoplastic versus normal T cells as both express similar levels of CD5. CARTresistant normal T cells are developed to be co-infused with the anti-T-NHL CART to ensure immunity during CART anti-tumor activity. CRISPR-Cas9 gene-editing is used to knock out CD5 in normal T cells thus making them invisible to CART5. A highly efficient CRISPR- 20 Cas9 gRNA (#4) was generated that can KO -95% of CD5 in normal T cells using an optimized CART expansion protocol. Data show that CD5 KO T cells were resistant to CART5 while WT T cells (CD5+) were potently killed within 24 hours. Manufacturing of CD5 KO normal T cells: CD5 KO normal T cells are developed using a highly efficient gRNA (#4) that is electroporated together with Cas9 protein 25 (ThermoFisher v2) using a Lonza 4D Nucleofector. CRISPR-Cas9 KO is performed on day 1, then cells are cultured at 30°C for 2 days to increase gene-editing, then activated with anti-CD3 / CD28 Dynabeads (beads: :1 T cells) and expanded until they reach a cell volume <300 fl. In vitro evaluation of CD5 KO normal T cell resistance to iCART5 killing: 30 Resistance of CD5 KO normal T cells to CART5 is tested in vitro by performing killing 2026223298   27 Aug 2026 assays (CFSE labeling of target T cells). Preliminary resultsshowed that CD5 KO confers resistance. Three additional T cell donors will be tested. In vivo evaluation of CD5 KO normal T cell resistance to iCART5 in an autologous xenograft model: NSG mice (8 mice / group) are engrafted with luciferase+ CD5 KO normal 5 T cells or WT, and after two days autologous iCART5 is injected. The effect of iCART5 on CD5 ko and WT normal T cells is assessed by bioluminescence. Once WT T cells are completely eliminated by iCART5 (luminescence) rimiducid is administered to deplete iCART5. Then WT T cells are reinjected to demonstrate that normal T cells can repopulate the host. Mice are bled weekly to assess CART expansion. 10          Evaluation of the role of CD5 KO on normal T cell functions: The role of CD5 KO is investigated in normal T cells by carefully studying T cell effector functions. After TCR-specific stimulation (anti-CD3 / CD28 beads) cytokine production (30-plex Luminex), proliferation (CFSE) and activation of CD5KO T cells vs. WT are measured. Also tested are whether CD5KO T cells proliferate and produce similarly to WT when exposed to common 15 infections. 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 for infectious agents) the minimum number of cells to be infused in relapsed or refractor (r / r) T-NHL patients is defined in order to ensure sufficient T cell immunity to the most common 20 infections. Testing the concurrent infusion of iCART5 and CD5 KO normal T cells in a phase 1 pilot clinical trial for patients with advanced T cell lymphoma: An Investigation New Drug (IND) package is developed and submitted to the FDA. A phase 1 clinical trial is started to test the anti-T-NHL CART approach in patients. The IND package is based on preliminary 25 results and further data from experiments described herein. Clinical trial protocol design: The phase 1 clinical trial includes patients with r / r T-NHL that are treated using a 3+3 protocol design. From a single apheresis, two products are generated using enriched T cells: #1. CRISPR-Cas9 CD5 KO normal T cells and #2. iCART5. The first product to be infused is the KO normal T cells followed the next day by 30 iCART5. The first cohort of patients receives lymphodepletion [cyclophosphamide (60mg / kg x 2 days) and fludarabine (25 mg / m2 x 5 days)] and product #1. If no dose-limiting toxicity 2026223298   27 Aug 2026 (DLT) is observed, Cohort 2 receives lymphodepletion, product #1 and l-5xl0e7 total iCART5 (product#2) over 3 days (10%, 30%, 60% of the total dose); l-5xl0e7 total CART5 is a suboptimal dose based on CART19 experiments in B-NHL8. If no DLT is observed in cohort 2, cohort 3 receives lymphodepletion, product #1 and l-5xl0e8 total CART5 (full 5 dose). Patient from cohort #1 will is allowed to proceed to cohort #2 if no toxicity is observed within the first 4 weeks. Based on tumor clearance (and maximum at month 6) the iCART5 cells are depleted using the dimerizing agent rimiducid (NCT02744287) to prevent possible long-term T cell toxicity. Preparation of IND package and FDA submission: Optimization of clinical-grade 10 manufacturing is run in collaboration with the Clinical Vaccine and Cell Production Facility (CVPF). The results of all described preclinical experiments together with the clinical trial protocol are formatted to fit an IND application. Extensive support for the IND preparation is available within the CCI and ACC. Patient enrollment and treatment: After successful submission of the IND and 15 approval from all the regulatory agencies, the phase 1 trial is started at the University of Pennsylvania, within the Lymphoma Program (Director: Dr. Stephen Schuster; Scientific Director: Dr. Marco Ruella). The Lymphoma program has a dedicated clinical research unit (CRU) with extensive experience managing early-stage studies. Dr. Ruella is the Principal Investigator of this trial with Dr. Carl June being the Scientific Protocol Advisor. 20 Manufacturing of the 2 products is performed at the CVPF. Correlative studies: Patient samples (peripheral blood) are analyzed at multiple time points (apheresis, day - 1,0, 7, 14, 28, 60, 90) to test CART expansion (qPCR and flow cytometry), CART phenotype (CyTOF), CART gene expression profiling (GEP) (NanoString, single-cell RNAseq 10X Genomics) and cytokine levels in the serum 25 (Luminex, 30-plex array). Additional studies are performed on tumor biopsies pre-treatment and post-treatment when available (RNAseq and Hyperion analysis of the tumor microenvironment). This trial will be a key milestone in the development of novel combined immunotherapies as it represents an innovative immunotherapeutic approach to treat T cell 30 non-Hodgkin lymphomas avoiding toxicity. Anti-CD5 CAR T cells kill tumor T cells but unavoidably also normal T cells due to similar CD5 expression. However, the strategy 2026223298   27 Aug 2026 described herein includes the co-infusion of normal T cells that have been knocked out for CD5, thereby ensuring T cell immunological protection during CART5 anti-tumor activity. CART5 cells are then depleted using a suicide system to ensure normal immunological reconstitution long term. This is one of the first CART trials for T-NHL and the only one 5 including a two-pronged approach addressing the issue of toxicity. T-NHL have a very poor prognosis, and there are currently no active immunotherapies available. Therefore the development of such an innovative strategy represents a vertical advance in the field of hematology and immunotherapy. Based on the clinical results of the phase 1 trials and the findings of the correlative studies this strategy can also be implemented to targeting multiple 10 targets at the same time to avoid antigen-loss escape (e.g., CART5+CART7) or combine CART with small molecules that can enhance CART-mediated killing. Example 3: Anti-CD2 CAR T cells (CART2) and CD2 knocked-out (KO) normal T cells A two-pronged immunotherapy approach is disclosed herein that includes anti-CD2 15   CAR T cells (CART2) and CD2 knocked-out (KO) normal T cells (FIG. 3). The 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 CART2 cells are depleted, in some cases by using a suicide gene (e.g. iCasp9). Guide RNAs were designed to knock out the CD2 gene (and CD5) using the 20 CRISPR / Cas9 system. CD2 was effectively knocked out 78% of the T cell population. Second generation anti-CD2 and anti-CD5 CARs (CART2 and CART5, respectively) were generated (FIG. 4). Knock out cells (CD2KO and CD5KO) were incubated with their corresponding CART cells (CART2 and CART5, respectively), stimulated, and population doublings measured (FIG. 7). Mock electroporated cells that did not contain gRNA were 25 used as a control for comparison. Without CD2 KO CART2 cells would not expand (FIG. 7). With KO CART2 and CART5 reach about 5-8 population doublings (FIG. 7). Jurkat cells were transduced with the different CAR2 constructs and with a GFP-NFAT reporter then co-cultured with CD2+ tumor cells (or controls) for 24 hours. The lead CART2 (C3043) shows increased NF AT activation (FIG. 14). 30         The CART expansion protocol was optimized CART2 and CART5 cells continued to expand up to 18 days when incubated with CD2KO or CD5 KO cells (FIG. 9). 2026223298   27 Aug 2026 Example 4: CART2 and CART5 Testing FIG. 6 illustrates the CD5 (or CD2, or CD7) KO manufacturing process and CRISPR-Cas9 KO efficiency. 5          The 6 different CAR2 and 6 CAR5 constructs were challenged in vitro by co culturing them with luciferase+ Jurkat cells (T-cell leukemia cell line). At 24 hrs, total killing was measured as relative reduction in luminescence. For CART2 only #3029, #3030 and #3043 show anti-tumor effect. (FIG. 10). For CART5, all the constructs demonstrated antitumor effects (FIG. 11). Lead CART candidates (CART2 C3043 and CART5 C3054) were 10 selected and tested . The effects of CD2 or CD5 knock-out on CART function was tested). CART2- and CART5-resistant T cells were successfully generated (. CD5 and CD2 were knocked-out in normal T cells. CART2 and CART5 activity against cutaneous T cell lymphoma was tested. Twenty-four hour killing assays were performed. CART2 cells were active against primary Sezary 15 cells (leukemic Cutaneous T Cell Lymphoma) and the HH Sezary cell line (FIG. 15). Also CART5 were active against HH cells (FIG. 15). In vivo efficacy of CART2 and CART5 was measured. NSG mice were engrafted with Luciferase+ Jurkat cells and mice were randomized to receive control T cells or CART2 or CART5 (IxlO6) at day 7. Mice were imaged weekly using the IVIS Xenogen Spectrum 20 and analyzed with Livingimage software. CART2 C3043 and CART5 C3054 were the most effective (FIG. 12). CART2 and CART5 were demonstrated to recognize normal T cells (autologous and allogeneic) and kill them (FIG. 16). It was also demonstrated that removal of the CAR target protects normal T cells from 25 CART killing (FIG. 17). CD5 KO but not WT normal T cells were resistant to CART5 killing. Normal resting T cells are recognized and killed by CART2 (FIG. 17, top) and CART5 (FIG. 17, bottom). Efficient KO of CD2 or CD5 from normal T cells using CRISPR-Cas9 led to resistance to CART2 or CART5 killing respectively (FIG. 17). CMV-specific T cells were present in CD2KO and CD5KO normal T cell products 30 (FIG. 18). CD2 and CD5 KO normal T cells maintained the ability to recognize CMV peptides and produce cytokines (FIG. 18; HLA-A-02:01 - CMVPP65 NLVPMVATV 2026223298   27 Aug 2026 dextramer (SEQ ID NO: 101); ICS after 4h exposure to CETF peptides. After secondary culture with CMV-peptide pulsed APC). Example 5: Anti-CD7 CAR T cells (CART7) and CD7 knocked-out (KO) normal T cells 5          A two-pronged immunotherapy approach is disclosed herein that includes anti-CD7 CAR T cells (CART7) and CD7 knocked-out (KO) normal T cells. The CART7 destroys T cell lymphoma (e.g. T-NHL) or T cell leukemia cells, but also kills normal T cells. Infusion of CD7 KO normal T cells provides CART-resistant T cell immunity until CART7 cells are depleted, in some cases by using a suicide gene (e.g. iCasp9). 10         Guide RNAs were designed to knock out the CD7 gene using the CRISPR / Cas9 system. CD7 was effectively knocked out 79% of the T cell population (FIG. 25). Six anti-CD7 CARs were generated (FIG. 25). Example 6: Dual specific CAR T cells 15          Two lentiviral constructs were generated that included CAR5 (C3054) and CAR2 (C3043) linked by a P2A sequence (FIG. 19). Gene expression was driven by an EFlalpha promoter. The CAR5 construct has 4-IBB costimulatory and CD3zeta signaling domains. Efficient knock-out of both CD2 and CD5 in normal T cells was demonstrated (FIGs. 20A-20B). 20 Example 6: CD5 KO enhances CART immunotherapy It was demonstrated that CD5 KO CART5s are more effective than CD5+ CART5s in vivo. CD5 KO increased CART5 anti-tumor efficacy (FIG. 21). In a Jurkat T-ALL xenograft model using NSG mice, CD5 KO CART5 (2xl06 cells / mouse) lead to complete long-term 25 responses and longer survival as compared to WT CART5 (FIG. 21). CD5 KO CART19 were also more effective than CD5+ CART19 in vivo. CD5 KO increased CART19 anti-tumor efficacy (FIG. 22). In aNALM6 B-ALL xenograft model CD5 KO CART 19 exhibited drastically higher tumor control as compared to WT CART 19 (FIG. 22). 2026223298   27 Aug 2026 CART5 and CART2 were also capable of targeting 20% of AML. CART2 cells were co-cultured with CD2+ AML cells and showed significant killing at 24 hours (FIGs. 23 A-23B). CART5 also targeted 100% of CLL and MCL. A cytotoxicity assay was performed 5 and demonstrated that CART5 cells can recognize and kill CD5+ MCL cell lines (Jeko-1 and Mino) (FIG. 24). These data demonstrate that knocking out CD5 enhances CART therapy when treating with an anti-CD5 CAR, or surprisingly when treating with a different CAR T cell (e.g. a CD 19 CART cell). 10 Other Embodiments The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any 15 single embodiment or in combination with any other embodiments or portions thereof. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing 20 from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A pharmaceutical composition comprising a first population of T cells comprising anexogenous nucleic acid molecule encoding a chimeric antigen receptor (CAR) and a mutated endogenous CD5 gene and a second population of T cells comprising a mutated endogenous CD5 gene,wherein the second population does not comprise the exogenous nucleic acid molecule encoding the CAR,wherein the first and second population of T cells comprising the mutated endogenous CD5 gene have a decrease in expression of endogenous CD5 protein.

2. The pharmaceutical composition of claim 1, wherein the mutated endogenous CD5 geneis a gene edited endogenous CD5 gene.

3. The pharmaceutical composition of claim 1, wherein the mutated endogenous CD5 geneis mutated using CRISPR.

4. The pharmaceutical composition of claim 1, wherein the decrease in expression ofendogenous CD5 protein is achieved using a CRISPR / Cas9 method.

5. The pharmaceutical composition of claim 4, wherein the CRISPR / Cas9 method utilizesan sgRNA comprising a nucleotide sequence of SEQ ID NO: 23.

6. The pharmaceutical composition of any one of claims 1-5, wherein the CAR encoded bythe exogenous nucleic acid molecule does not comprise an antigen binding domain that binds to CD5.

7. The pharmaceutical composition of any one of claims 1-6, wherein the CAR comprisesan antigen binding domain, a transmembrane domain, and an intracellular domain,2026223298   27 Aug 2026wherein the transmembrane domain comprises a transmembrane domain derived from the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154; andwherein the intracellular domain comprises one or more intracellular signaling domains selected from CD3-zeta, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

8. The pharmaceutical composition of any one of claims 1-6, wherein the CAR comprisesan antigen binding domain capable of binding an antigen selected from the group consisting of CD5, CD19, CD2, CD7, a tumor-specific antigen (TSA), a tumor associated antigen (TAA), a glioma-associated antigen, carcinoembryonic antigen (CEA), p-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, Ras, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, HPV antigen E6, HPV antigen E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 199, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7- Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

9. The pharmaceutical composition of claims 7 or 8, wherein the antigen binding domain ofthe CAR binds to CD5 and comprises: i) a heavy chain variable domain (VH) comprising a HCDR1 comprising an amino acid sequence of SEQ ID NO: 83, a HCDR2 comprising an amino acid sequence of SEQ ID NO: 84, and a HCDR3 of SEQ ID NO: 85; and ii) a light chain variable domain (VL) comprising a LCDR1 comprising an amino acid sequence of SEQ ID NO:2026223298   27 Aug 202686, a LCDR2 comprising an amino acid sequence of SEQ ID NO: 87, and a LCDR3 comprising an amino acid sequence of SEQ ID NO: 88.

10. The pharmaceutical composition of any one of claims 7-9, wherein the antigen binding domain of the CAR binds to CD5 and comprises a heavy chain variable domain (VH)and a light chain variable domain (VL); wherein the VH comprises an amino acid sequence having at least 90% identity to an amino acid sequence of SEQ ID NO: 81 and comprises a HCDR1 comprising an amino acid sequence of SEQ ID NO: 83, a HCDR2 comprising an amino acid sequence of SEQ ID NO: 84, and a HCDR3 of SEQ ID NO: 85; and wherein the VL an amino acid sequence having at least 90% identity to an amino acid sequence of SEQ ID NO: 82 and comprises a LCDR1 comprising an amino acid sequence of SEQ ID NO: 86, a LCDR2 comprising an amino acid sequence of SEQ ID NO: 87, and a LCDR3 comprising an amino acid sequence of SEQ ID NO: 88.

11. The pharmaceutical composition of any one of claims 7-9, wherein the antigen binding domain of the CAR binds to CD5 and comprises a heavy chain variable domain (VH) and a light chain variable domain (VL); wherein the VH comprises an amino acid sequence having at least 95% identity to an amino acid sequence of SEQ ID NO: 81 and comprises a HCDR1 comprising an amino acid sequence of SEQ ID NO: 83, a HCDR2 comprising an amino acid sequence of SEQ ID NO: 84, and a HCDR3 of SEQ ID NO: 85; and wherein the VL an amino acid sequence having at least 95% identity to an amino acid sequence of SEQ ID NO: 82 and comprises a LCDR1 comprising an amino acid sequence of SEQ ID NO: 86, a LCDR2 comprising an amino acid sequence of SEQ ID NO: 87, and a LCDR3 comprising an amino acid sequence of SEQ ID NO: 88.

12. The pharmaceutical composition of claim 7 or 8, wherein the antigen binding domain of the CAR binds to CD5 and comprises a heavy chain variable domain (VH) comprising a HCDR1, HCDR2, and HCDR3 of the sequence shown in SEQ ID NO: 75 and comprises an amino acid sequence having at least 90% identity to an amino acid sequence of SEQ ID NO: 75, and a light chain variable domain (VL) comprising a LCDR1, LCDR2, and LCDR3 of the2026223298   27 Aug 2026sequence shown in SEQ ID NO: 76 and comprises an amino acid sequence having at least 90% identity to an amino acid sequence of SEQ ID NO: 76.

13. The pharmaceutical composition of claim 12, wherein the VH comprises an amino acid sequence having at least 95% identity to an amino acid sequence of SEQ ID NO: 75, and the VL comprises an amino acid sequence having at least 95% identity to an amino acid sequence of SEQ ID NO: 76.

14. The pharmaceutical composition of claim 12 or claim 13, wherein the VH comprises the amino acid sequence of SEQ ID NO: 75, and the VL comprises the amino acid sequence of SEQ ID NO: 76.

15. The pharmaceutical composition of any one of claims 12-14, wherein the antigen binding domain of the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 73 or SEQ ID NO: 74.

16. The pharmaceutical composition of any one of claims 12-15, wherein the antigen bindingdomain of the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 73 or SEQ ID NO: 74.

17. The pharmaceutical composition of any one of claims 12-16, wherein the antigen binding domain of the CAR comprises an amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74.

18. The pharmaceutical composition of claim 7 or 8, wherein the antigen binding domain of the chimeric antigen receptor binds to CD5 and comprises a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of:i) SEQ ID NOs: 31-36, respectively;ii) SEQ ID NOs: 43-48, respectively;iii) SEQ ID NOs: 53-58, respectively;iv) SEQ ID NOs: 65-70, respectively;v) SEQ ID NOs: 83-88, respectively; or2026223298   27 Aug 2026vi) SEQ ID NOs: 95-100, respectively.

19. The pharmaceutical composition of claim 18, wherein the antigen binding domain of the CAR comprises an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 39, 40, 50, 61, 62, 79, 80, 91, and 92.

20. The pharmaceutical composition of any one of claims 12-17, wherein the antigen binding domain of the CAR is encoded for by a nucleic acid sequence having at least 80% identity to SEQ ID NO: 13.

21. The pharmaceutical composition of any one of claims 5-20, wherein the transmembrane domain of the CAR comprises an amino acid sequence of SEQ ID NO: 15.

22. The pharmaceutical composition of any one of claims 5-21, wherein the intracellular domain of the CAR comprises a 4-1BB signaling domain comprising an amino acid sequence of SEQ ID NO: 20 and a CD3-zeta signaling domain comprising an amino acid sequence of SEQ ID NO: 21.

23. The pharmaceutical composition of any one of claims 5-21, wherein the CAR further comprises a hinge sequence comprising an amino acid sequence of SEQ ID NO: 17.

24. The pharmaceutical composition of any one of claims 12-17, wherein the CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 71 or SEQ ID NO: 72.

25. The pharmaceutical composition of any one of claims 12-17, wherein the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 71 or SEQ ID NO: 72.

26. The pharmaceutical composition of any one of claims 12-17, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 72.2026223298   27 Aug 202627. The pharmaceutical composition of any one of claims 7-11, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 60,, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 89, or SEQ ID NO: 90.

28. The pharmaceutical composition of any one of claims 1-27, wherein the chimeric antigen receptor further comprises a suicide gene.

29. The pharmaceutical composition of claim 28, wherein the suicide gene is iCaspase9.

30. A method of treating a cancer in a subject in need thereof, the method comprisingadministering to the subject the pharmaceutical composition of any one of claims 1-29.

31. The method of claim 30, wherein the cancer is selected from the group consisting of a T cell lymphoma, a T cell leukemia, acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

32. A chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that binds to CD5, wherein the antigen binding domain comprises i) a VH comprising a HCDR1, a HCDR2, and a HCDR3 of the sequence shown in SEQ ID NO: 75, and ii) a VL comprising a LCDR1, a LCDR2, and a LCDR3 of the sequence shown in SEQ ID NO: 76.

33. The CAR of claim 32, wherein the VH comprises an amino acid sequence having at least 90% identity to an amino acid sequence of SEQ ID NO: 75, and the VL comprises an amino acid sequence having at least 90% identity to an amino acid sequence of SEQ ID NO: 76.

34. The CAR of claim 32, wherein the VH comprises an amino acid sequence having at least 95% identity to an amino acid sequence of SEQ ID NO: 75, and the VL comprises an amino acid sequence having at least 95% identity to an amino acid sequence of SEQ ID NO: 76.2026223298   27 Aug 202635. The CAR of claim 32, wherein the VH comprises the amino acid sequence of SEQ ID NO: 75, and the VL comprises the amino acid sequence of SEQ ID NO: 76.

36. The CAR of any one of claims 32-35, wherein the antigen binding domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 73 or SEQ ID NO: 74.

37. The CAR of any one of claims 32-36, wherein the antigen binding domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 73 or SEQ ID NO: 74.

38. The CAR of any one of claims 32-37, wherein the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74.

39. The chimeric antigen receptor of any one of claims 32-38, wherein the antigen binding domain of the CAR is encoded for by a nucleic acid sequence having at least 80% identity to SEQ ID NO: 13.

40. A nucleic acid molecule encoding the CAR of any one of claims 32-38.

41. A vector comprising the nucleic acid molecule of claim 40.

42. A cell comprising the nucleic acid molecule of claim 40 or the vector of claim 41.

Images