CD2 chimeric switch receptors and uses thereof for adoptive cell transfer immunotherapy

Abstract

The present disclosure provides chimeric switch receptors comprising a CD2 intracellular domain (e.g., a PD-1:CD2 switch receptor) and modified immune cells (e.g., T cells) comprising the switch receptor and a chimeric antigen receptor (CAR) or T cell receptor (TCR). Also provided are methods and uses of the immune cell for treating cancer. Related nucleic acids, vectors, and pharmaceutical compositions are also provided.

Description

[0001] Attorney Docket No. 046483 -7478WO 1(04048)

[0002] CD2 CHIMERIC SWITCH RECEPTORS AND USES THEREOF FOR ADOPTIVE CELL TRANSFER IMMUNOTHERAPY

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 707,411, filed October 15, 2024, which application is hereby incorporated herein by reference in its entirety.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] This application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML file, created on Oct. 13, 2025, is named 046483-7478WO1 Sequence Listing.xml and is 24,576 bytes in size

[0007] BACKGROUND OF THE INVENTION

[0008] Chimeric antigen receptor (CAR) T cell therapy is a potentially curative treatment modality for patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphoma (NHL), and multiple myeloma (Schuster, et al., New England Journal of Medicine. 2017;377(26):2545-2554; Schuster, et al., New England Journal of Medicine. 2019;380(l):45- 56; Neelapu, et al., New England Journal of Medicine. 2017;377(26):2531- 2544; Maude, et al., New England Journal of Medicine. 2018;378(5):439-448; Munshi, et al., New England Journal of Medicine. 2021;384(8):705-716; Rodriguez, et al., N Engl J Med. 2023;388(l 1): 1002-1014). However, CAR T cells analogously engineered to target T cell- derived leukemias and lymphomas have not yet transformed current treatment practices. Several obstacles related to T cell immunobiology have restricted CAR T cell efficacy against T-cell neoplasms (Angelos, et al., Transplant Cell Ther. 2024;30(2): 171-186; Ghilardi, et al, Br J Haematol. 2021;193(3):449-465). Paramount is the lack of a singular and uniform T-cell antigen that discriminates neoplastic from healthy, endogenous T-cells. Although potential specific targets have been recently explored, initial clinical feasibility and activity are limited given issues with variable antigen expression amongst T cell neoplasm subsets and lack of CAR T cell in vivo expansion (Maciocia, et al., Nat Med. 2017;23(12): 1416-1423; Maciocia, et al., Blood. Attorney Docket No. 046483 -7478WO 1(04048)

[0009] 2022; 140(1 ):25-37; Cwynarski, et al., Blood. 2022;140(Supplement 1)40316-10317; Sanchez- Martinez, et al., Blood. 2019;133(21):2291-2304; Scarfo, et al, Blood. 2018;132(14):1495- 1506). Furthermore, administrating CAR T cells against heterogeneously expressed tumor antigens has a high potential for disease relapse due to an inability to eliminate all malignant clones. There is a need in the art for enhancing the anti-tumor efficacy of adoptive cell transfer immunotherapies in order to improve the clinical outcome of patients. The present invention addresses this need.

[0010] SUMMARY OF THE INVENTION

[0011] In some aspects, the invention provides a chimeric switch receptor comprising:

[0012] (i) an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain;

[0013] (ii) a transmembrane domain; and

[0014] (iii) an intracellular domain comprising a CD2 intracellular signaling domain. In some embodiments, the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T- lymphocyte- Associated Protein 4 (CTLA4); and the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand-1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte- Associated Protein 4 (CTLA4).

[0015] In some embodiments, the transmembrane domain is a CD2 transmembrane domain.

[0016] In some embodiments, the chimeric switch receptor comprises:

[0017] (i) a PD1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain; or

[0018] (ii) an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain. Attorney Docket No. 046483 -7478WO 1(04048)

[0019] In some embodiments, the anti -checkpoint inhibitor antigen binding domain is a singlechain variable fragment (scFv).

[0020] In some embodiments, the chimeric switch receptor comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 1.

[0021] In some embodiments, the chimeric switch receptor comprises:

[0022] (i) a leader sequence comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 2;

[0023] (ii) a PD-1 LBD comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 3;

[0024] (iii) a CD2 transmembrane domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 4; and / or

[0025] (iv) a CD2 intracellular signaling domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 5.

[0026] In some aspects, the invention provides a nucleic acid comprising a nucleotide sequence encoding the chimeric switch receptor of any one of the preceding embodiments. Attorney Docket No. 046483 -7478WO 1(04048)

[0027] In some aspects, the invention provides a vector comprising the nucleic acid comprising a nucleotide sequence encoding the chimeric switch receptor of any one of the preceding embodiments.

[0028] In some embodiments, the vector is a retroviral vector or a lentiviral vector.

[0029] In some aspects, the invention provides a nucleic acid comprising:

[0030] (i) a first nucleotide sequence encoding a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and

[0031] (ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

[0032] In some embodiments, the nucleic acid further comprises a self-cleaving peptide sequence between the first nucleotide sequence and the second nucleotide sequence.

[0033] In some embodiments, the self-cleaving peptide sequence is a P2A sequence.

[0034] In some embodiments, the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T- lymphocyte- Associated Protein 4 (CTLA4); and the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand-1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte- Associated Protein 4 (CTLA4).

[0035] In some embodiments, the transmembrane domain of the chimeric switch receptor is a CD2 transmembrane domain.

[0036] In some embodiments, the chimeric switch receptor comprises:

[0037] (i) a PD1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain; or

[0038] (ii) an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain. Attorney Docket No. 046483 -7478WO 1(04048)

[0039] In some embodiments, the chimeric switch receptor comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 1.

[0040] In some embodiments, the chimeric switch receptor comprises:

[0041] (i) a leader sequence comprising an amino acid sequence having at least 80%, at least

[0042] 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

[0043] 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least

[0044] 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least

[0045] 99%, or 100% sequence identity to SEQ ID NO: 2;

[0046] (ii) a PD-1 LBD comprising an amino acid sequence having at least 80%, at least

[0047] 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

[0048] 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least

[0049] 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least

[0050] 99%, or 100% sequence identity to SEQ ID NO: 3;

[0051] (iii) a CD2 transmembrane domain comprising an amino acid sequence having at least

[0052] 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least

[0053] 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least

[0054] 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0055] 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; and / or

[0056] (iv) a CD2 intracellular signaling domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 5.

[0057] In some embodiments, the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain.

[0058] In some embodiments, the CAR or the TCR targets a tumor antigen.

[0059] In some embodiments, the tumor antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, Attorney Docket No. 046483 -7478WO 1(04048)

[0060] CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE- A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D- Ligands (MIC -A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, R0R1, R0R2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

[0061] In some embodiments, the anti -checkpoint inhibitor antigen binding domain of the chimeric switch receptor comprises a single-chain variable fragment (scFv).

[0062] In some embodiments, the tumor antigen binding domain of the CAR comprises a singlechain variable fragment (scFv).

[0063] In some embodiments, the intracellular domain of the CAR comprises a costimulatory domain of CD28, a costimulatory domain of 4- IBB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

[0064] In some embodiments, the CAR comprises an anti-CD19 antigen binding domain, a transmembrane domain, a 4- IBB costimulatory domain, and a CD3 zeta signaling domain.

[0065] In some aspects, the invention provides a vector comprising the nucleic acid described herein.

[0066] In some embodiments, the vector is a retroviral vector or a lentiviral vector.

[0067] In some aspects, the invention provides a modified immune cell, or precursor cell thereof, comprising the chimeric switch receptor described herein, the nucleic acid described herein, and / or the vector described herein.

[0068] In some embodiments, the cell is a T cell, an autologous cell, a human cell, or any combination thereof.

[0069] In some aspects, the invention provides a modified immune cell or precursor cell thereof, comprising:

[0070] (i) a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a Attorney Docket No. 046483 -7478WO 1(04048) transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and

[0071] (ii) a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

[0072] In some embodiments, the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T- lymphocyte-Associated Protein 4 (CTLA4); and the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand- 1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte- Associated Protein 4 (CTLA4).

[0073] In some embodiments, the transmembrane domain of the chimeric switch receptor is a CD2 transmembrane domain.

[0074] In some embodiments, the chimeric switch receptor comprises:

[0075] (i) a PD1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain; or

[0076] (ii) an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

[0077] In some embodiments, the chimeric switch receptor comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 1.

[0078] In some embodiments, the chimeric switch receptor comprises:

[0079] (i) a leader sequence comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

[0080] 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least

[0081] 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least

[0082] 99%, or 100% sequence identity to SEQ ID NO: 2; Attorney Docket No. 046483 -7478WO 1(04048)

[0083] (ii) a PD-1 LBD comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 3;

[0084] (iii) a CD2 transmembrane domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 4; and / or

[0085] (iv) a CD2 intracellular signaling domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 5.

[0086] In some embodiments, the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain.

[0087] In some embodiments, the CAR or the TCR targets a tumor antigen.

[0088] In some embodiments, the tumor antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE- A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D- Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

[0089] In some embodiments, the anti-checkpoint inhibitor antigen binding domain of the chimeric switch receptor comprises a single-chain variable fragment (scFv). Attorney Docket No. 046483 -7478WO 1(04048)

[0090] In some embodiments, the tumor antigen binding domain of the CAR comprises a singlechain variable fragment (scFv).

[0091] In some embodiments, the intracellular domain of the CAR comprises a costimulatory domain of CD28, a costimulatory domain of 4- IBB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

[0092] In some embodiments, the CAR comprises an anti-CD19 antigen binding domain, a transmembrane domain, a 4- IBB costimulatory domain, and a CD3 zeta signaling domain.

[0093] In some embodiments, the modified immune cell or precursor cell thereof comprises an expression vector comprising a first nucleotide sequence encoding the chimeric switch receptor and a second nucleotide sequence encoding the CAR or the TCR.

[0094] In some embodiments, the expression vector is a lentiviral vector or a retroviral vector.

[0095] In some embodiments, the modified immune cell or precursor cell thereof is a T cell, an autologous cell, a human cell, or any combination thereof.

[0096] In some aspects, the invention provides a pharmaceutical composition comprising a population of the modified immune cell or precursor cell thereof described herein and a pharmaceutically acceptable carrier.

[0097] In some embodiments, the pharmaceutical composition described herein is for use in a method of treating cancer in a subject in need thereof.

[0098] In some aspects, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of modified immune cells or precursor cells thereof, wherein the modified immune cells or precursor cells thereof comprise:

[0099] (i) a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and

[0100] (ii) a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

[0101] In some embodiments, the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Attorney Docket No. 046483 -7478WO 1(04048)

[0102] Immunoreceptor with Ig and ITIM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T- lymphocyte- Associated Protein 4 (CTLA4); and the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand- 1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte- Associated Protein 4 (CTLA4).

[0103] In some embodiments, the transmembrane domain of the chimeric switch receptor is a CD2 transmembrane domain.

[0104] In some embodiments, the chimeric switch receptor comprises:

[0105] (i) a PD1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain; or

[0106] (ii) an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

[0107] In some embodiments, the chimeric switch receptor comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 1.

[0108] In some embodiments, the chimeric switch receptor comprises:

[0109] (i) a leader sequence comprising an amino acid sequence having at least 80%, at least

[0110] 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

[0111] 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least

[0112] 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least

[0113] 99%, or 100% sequence identity to SEQ ID NO: 2;

[0114] (ii) a PD-1 LBD comprising an amino acid sequence having at least 80%, at least

[0115] 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

[0116] 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least

[0117] 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least

[0118] 99%, or 100% sequence identity to SEQ ID NO: 3;

[0119] (iii) a CD2 transmembrane domain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least Attorney Docket No. 046483 -7478WO 1(04048)

[0120] 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 99%, or 100% sequence identity to SEQ ID NO: 4; and / or

[0121] (iv) a CD2 intracellular signaling domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 5.

[0122] In some embodiments, the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain.

[0123] In some embodiments, the CAR or the TCR targets a tumor antigen.

[0124] In some embodiments, the tumor antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypi can-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE- A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D- Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

[0125] In some embodiments, the anti-checkpoint inhibitor antigen binding domain of the chimeric switch receptor comprises a single-chain variable fragment (scFv).

[0126] In some embodiments, the tumor antigen binding domain of the CAR comprises a singlechain variable fragment (scFv).

[0127] In some embodiments, the intracellular domain of the CAR comprises a costimulatory domain of CD28, a costimulatory domain of 4- IBB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

[0128] In some embodiments, the CAR comprises an anti-CD19 antigen binding domain, a transmembrane domain, a 4- IBB costimulatory domain, and a CD3 zeta signaling domain. Attorney Docket No. 046483 -7478WO 1(04048)

[0129] In some embodiments, the modified immune cells or precursor cells thereof comprise an expression vector comprising a first nucleotide sequence encoding the chimeric switch receptor and a second nucleotide sequence encoding the CAR or the TCR.

[0130] In some embodiments, the expression vector is a lentiviral vector or a retroviral vector.

[0131] In some embodiments, the modified immune cells or precursor cells thereof are T cells, autologous cells, human cells, or any combination thereof.

[0132] In some embodiments, the subject is a human.

[0133] In some embodiments, the cancer is selected from the group consisting of a B-cell malignancy (e.g., a B-cell lymphoma or a leukemia), lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophagus cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, and renal cancer.

[0134] BRIEF DESCRIPTION OF THE DRAWINGS

[0135] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0136] FIGs. 1A - IF show that CD2 is required for optimal CAR T cell efficacy. FIG. 1A: Percent cytotoxicity of untransduced (Mock UTD), CD2KO UTD, CD2WT CART 19, or CD2KO CART19 cells against Nalm6 after 72 hours as continuously measured using a CellCyte Live Cell Analyzer (E:T ratio = 0.125: 1). Significance determined by ANOVA with Tukey post- hoc multiple comparisons test; *** p < 0.001 relative to CD2KO CART 19. FIG. IB: Schema of CART19 treatment. NSG mice were intravenously engrafted with 1.0 x 106Nalm6 and subsequently treated with 1.0 x 106untransduced T cells (Mock UTD), CD2KO UTD cells, 1.0 x 106CAR-positive CD2WT CART19, or 1.0 x 106CAR-positive CD2KO CART19 cells seven days after tumor injection. FIG. 1C: Total BLI of each individual mouse treated post T cell infusion is shown. Solid lines represent the median of each cohort. Mice were monitored either Attorney Docket No. 046483 -7478WO 1(04048) until death due to any cause or euthanized at the predetermined humane endpoint (Total Flux = 1011photons / sec). Significance determined by paired t-test between CD2KO CART19 and CD2WT CART19 at each time point; *** p < 0.001, ** p < 0.01, * p < 0.05. FIG. ID: Overall survival of composite NSG mice treated with Mock UTD, CD2KO UTD, CD2WT CART19, or CD2KO CART19 cells. Wilcox rank-sum test was performed for all survival curve comparison between CD2WT CART19 and CD2KO CART19; *p < 0.05. FIG. IE: Top: Representative images of activated CD2WT CART 19 (top row) or CD2KO CART 19 cell (bottom row) immune synapses by planar bilipid immunofluorescence microscopy against biotinylated CD 19. pCD3^ (magenta) is used as a marker for CAR molecules, which overlaps CD 19 protein (green) to reflect a CART19 immune synapse. Bottom'. Quantification of CD2 and F-actin localization to the CD19:CART19 immune synapses. Each data point represents an individual synapse. Significance determined by student’s t-test; **** p < 0.0001. FIG. IF: Quantification of Mock UTD, CD2KO UTD, CD2WT CART 19, and CD2KO CART 19 binding avidity to Nalm6 after a 15-minute co-culture using the LUMICKS z-Movi Cell Avidity Analyzer, one representative run shown.

[0137] FIGs. 2A - 2E show that CD2 loss alters CART19 activity. FIG. 2A: Population doublings of two, independent, healthy T cell donor expansions of CART19 with or without CD2 and either a 4-lBBz (CART19-BBz) or CD28z (CART19-28z) costimulatory domain. FIG. 2B: Replicative bioluminescence imaging of leukemia burden engrafted with 1.0 x 106Nalm6 (top) and overall survival (bottom) of composite NSG mice treated with 1.0 x 106Mock UTD, CD2KO UTD, CD2-wild type (CD2WT) CART19-BBz, and CD2KO CART19-BBz cells from a separate, independent healthy T cell donor. n= 3-6 mice per group. FIG. 2C: Body mass of each NSG mouse in both donor cohorts. FIG. 2D: Serum cytokine quantification of NSG mouse plasma fractionated from peripheral blood at Day 7 post Mock UTD, CD2KO UTD, CD2WT CART19, and CD2KO CART19 treatment, as determined by Luminex multiplex assay. FIG. 2E: Bioluminescence imaging of leukemia burden engrafted with 1.0 x 106Nalm6 (top) and overall survival (bottom) of composite NSG mice treated with 1.0 x 106Mock UTD, CD2KO UTD, CD2-wild type (CD2WT) CART19-28z, and CD2KO CART19-28z cells. Total BLI of each individual mouse treated (dashed lines) post T cell infusion is shown. Solid lines represent the median of each cohort. Wilcox rank-sum test was performed between CD2WT CART 19 and CD2KO CART19 for all survival curve comparisons; ** p < 0.01, * p < 0.05. Attorney Docket No. 046483 -7478WO 1(04048)

[0138] FTGs. 3 A - 3E shows single cell RNA sequencing analysis of in vivo derived CD2KO CART19. FIG. 3A: Left: NSG mice were subcutaneously engrafted with 5.0 x 106OCI-Lyl8 (diffuse large B cell lymphoma) cells on day -10 and intravenously injected with 4.0 x 106Mock or CD2KO CART 19 cells on day 0. Right. Bioluminescence imaging of tumor burden in each NSG mouse (n= 4-8 mice per group) engrafted with the OCI-Lyl8 cell line. Bolded line represents the mean of each group. FIG. 3B: UMAP of all clusters amongst Mock CART 19 and CD2KO CART 19 populations. Clusters 6 (red) and 7 (orange) represent CD2KO T cell-specific clusters. FIG. 3C: Cluster-specific genes differentially regulated between Clusters 6 and 7, as compared to all other conserved clusters. FIG. 3D: Gene set enrichment analysis identifies T cell activation as an underrepresented pathway within clusters 6 and 7. NES = normalized enrichment score. FIG. 3E: List of cluster-specific genes driving the association to reduced T cell activation. Cluster comparisons were analyzed by ANOVA with Tukey post-hoc multiple comparisons test **** P < 0.0001, *** p < 0.001, ** p < 0.01.

[0139] FIGs. 4A - 4C show ancillary single cell RNA sequencing analyses of in vivo derived CD2KO CART 19. FIG. 4A: Immunophenotype of Mock UTD (left) and Mock CART 19 (middle) T cells prior to injection into NSG mice, as correlated with FIG. 3A. To ensure complete CD2 deficiency for single cell analyses, CD2KO CART19 cells were additionally FACS-sorted based on CD2 expression prior to injection (right). FIG. 4B: Cell type annotations defined by colors indicating different T and natural killer (NK) cell types in the dataset. FIG. 4C: Normalized expression levels of CI)3f CD4, and CD8A across all analyzed cells.

[0140] FIGs. 5A - 51 show that CD2:CD58 signaling axis influences CAR T cell response. FIG. 5A: Scatterplot of normalized MAGeCK beta scores representing enriched and depleted sgRNAs utilized in a genome-wide knock-out screen of Brunello library-edited Nalm6 cells and CART 19, as previously described (Singh, et al., Cancer Discov. 2020;10(4):552-567). FIG. 5B: Overall survival of 17 patients with diffuse large B cell lymphoma (DLBCL) enrolled to UPCC13413 (NCT02030834) and stratified based on CD58 expression, as determined by bulk tumor RNA sequencing collected prior to CART19 administration. Low CD58: Lowest 1 / 3 of patients based on CD58 gene expression, High CD58: Upper 2 / 3 of patients based on CD58 gene expression. FIG. 5C: Left. CD58 immunohistochemistry h-score ofB cell lymphoma biopsies obtained either pre-CART19 treatment (n = 27 unique patients) or post-CART19 treatment (n = 23 unique patients). B cell lymphoma diagnoses included DLBCL (n = 11), follicular lymphoma (n = 10), Attorney Docket No. 046483 -7478WO 1(04048) or other aggressive B cell lymphoma, including marginal zone lymphoma, grey zone lymphoma, and primary mediastinal B cell lymphoma (n=6). Patients were treated either with tisagenlecleucel (tisa-cel, n=22) or axicabtagene ciloleucel (axi-cel, n=5). Middle-. H-scores of Long CR: Pre-CART19 biopsy of patients with a complete remission (CR) exceeding 1 year after CART 19 treatment, Short CR: Pre-CART19 biopsy of patients with a CR less than 1 year following CART19 treatment, Refractory / Relapsed: Pre-CART19 biopsy of patients refractory or relapsed within 90 days of CART 19 treatment. Right. Duration of complete responses stratified by pre-CART19 biopsy H-score. FIG. 5D: Left: Change in CD58 h-score (delta h- score) in patients where paired pre- and post-CART19 biopsies were available. Relapsed (R) refers to disease progression following a documented complete response and refractory (Rf) refers to failure to achieve a documented complete response. Right. Representative images of pre-CART19 (inset, top) and post-CART19 relapse (inset, bottom) of CD58 immunohistochemistry staining of a patient with DLBCL with relapsed disease after CART 19 treatment. FIG. 5E: Percent cytotoxicity of CART19 cells against OCI-Lyl8 and Nalm6 cells with (CD58WT) and without (CD58KO) CD58 expression across a range of effector-to-target cell ratios after 48 h coculture. Groups compared using ANOVA with Tukey post-hoc multiple comparisons test relative to CD58WT for each cell type; ** p < 0.01, * p < 0.05. FIG. 5F: Quantification of untransduced (UTD) or CART19 binding avidity to CD58WT and CD58KO Nalm6 after 15 minutes co-culture on the LUMICKS z-Movi Cell Avidity Analyzer. FIG. 5G: Schema of a PD-1 :CD2 switch receptor in CART19. FIG. 5H: Representative flow cytometry plots of CART19 (top panels) and PD-1 :CD2 switch receptor CART19 (bottom panels) with endogenous CD2KO at the end of expansion and following one freeze / thaw cycle. FIG. 51: Representative flow cytometry plots of CART 19 (top panels) and PD-LCD2 switch receptor CART 19 (bottom panels) with endogenous CD2KO at the end of expansion and following one freeze / thaw cycle.

[0141] FIGs. 6A - 6C relate to CD2:CD58 signaling axis rescue. FIG. 6A: Bulk RNA- sequencing of infusion products manufactured from 28 patients (13 follicular lymphoma, 15 diffuse large B cell lymphoma) treated with investigational, 4-lBB-based CART 19 (CTL019, NCT02030834), separated by best overall response. Response: complete response (CR) or partial response (PR) post-CTL019 treatment; No response = Neither CR nor PR post-CTL019 treatment. Groups compared using student’s t-test. FIG. 6B: Representative immunophenotype Attorney Docket No. 046483 -7478WO 1(04048) of Nalm6 and OCI-Lyl 8 cells with and without CD58 knock-out. FIG. 6C: Top-. Construct maps of a PD-l:CD2-intracellular signaling domain switch receptor integrated with a P2A selfcleaving peptide and CART19.BBz. Bottom-. Population doublings, as calculated by normalized viable cell count relative to the total number of cells initially electroporated, of each product cohort derived from a single healthy T cell donor.

[0142] FIGs. 7A - 7H show that CD2 is required for optimal CAR T-cell efficacy. FIG. 7A: Total fluorescence intensity of CD2WTUTD, CD2KOUTD, CD2WTCART 19, or CD2KOCART19 cells against Nalm6 after 72 hours as continuously measured using a CellCyte Live Cell Analyzer (E:T ratio = 0.125: 1). Significance determined by ANOVA with Tukey post-hoc multiple comparisons test; *** p < 0.001 relative to CD2KOCART19. FIG. 7B: Total BLI of each individual mouse treated post-T-cell infusion is shown. NSG mice were intravenously engrafted with 1.0 x 106Nalm6 and subsequently treated with 1.0 x 106CD2WTUTD, CD2KOUTD cells, 1.0 x 106CAR-positive CD2WTCART19, or 1.0 x 106CAR-positive CD2KOCART19 cells seven days after tumor injection. Solid lines represent the median of each cohort. Mice were monitored either until death due to any cause or euthanized at the predetermined humane endpoint (Total Flux = 1011photons / sec). Significance determined by paired t-test between CD2KOCART19 and CD2WTCART19 at each time point; ***p < 0.001, **p < 0.01, *p

[0143] < 0.05. FIG. 7C: Overall survival of composite NSG mice treated with CD2WTUTD, CD2KOUTD, CD2WTCART19, or CD2KOCART19 cells (mOS: CD2WTUTD: 14 days, CD2KOUTD: 14 days, CD2KOCART19: 28 days, CD2WTCART19: 38 days). Wilcox rank-sum test was performed for all survival curve comparison between CD2WTCART19 and CD2KOCART19; *p

[0144] < 0.05. FIG. 7D: Representative images of immune synapses formed by CD2WTCART19 (top) and CD2KOCART 19 cells (bottom), visualized by planar lipid bilayer immunofluorescence microscopy against biotinylated CD 19. pCD3(^ (magenta) marks CAR molecules and colocalizes with CD19 (green), delineating the CART19 synapse architecture. The experiment is designed to study the immune synapse from a top-down perspective, and what is shown is the imagine footprint of a single CAR-T cell engaging the bilayer. FIG. 7E: Quantification of CD2 and F- actin recruitment to the CD19:CART19 immune synapses. Each data point represents an individual synapse. Significance was assessed by Student’s t-test; ****p < 0.0001. FIG. 7F: Representative images of immunological synapses formed by CD2WTCART 19 (top) and CD2KOCART19 (bottom), stained for, in order: NALM6 GFP, CD5, pCD3(j, and the composite overlay. Attorney Docket No. 046483 -7478WO 1(04048)

[0145] Images were acquired using a Leica Stellaris confocal microscope with a 63x / l .4 oil immersion objective. FIG. 7G: Quantification of pCD3(^ signal length in immune synapses showed in (FIG. 7F). Each data point represents an individual synapse. Statistical analysis was performed using a Mann-Whitney test; ****p < 0.0001. FIG. 7H: Quantification of CD2WTUTD, CD2KOUTD, CD2W TCART19, and CD2KOCART19 binding avidity to Nalm6 after a 15-minute co-culture using the LUMICKS z-Movi Cell Avidity Analyzer, one representative run shown. Upon application of a 1000 pN acoustic force, CD2WTCAR19 T cells exhibited greater avidity toward target cells compared to CD2KOCAR19 T cells, as determined by Student’s t-test (p < 0.05).

[0146] FIGs. 8A - 8G relate to single-cell RNA sequencing analysis of in vivo-derived CD2KOCART19. FIG. 8A: NSG mice were subcutaneously engrafted with 5.0 x 106OCI-Lyl8 (diffuse large B-cell lymphoma) cells on day -10, followed by intravenous injection of 4.0 x io6Mock or CD2KOCART19 cells on day 0. FIG. 8B Median of the bioluminescence imaging of tumor burden in NSG mice (n = 8 for CD2WTCART19, n = 6 for CD2KOCART19) engrafted with OCI-Lyl8 cells. FIG. 8C: Unsupervised clustering using UMAP of all cells, grouped by experimental condition (left) or by T cell subset as defined via the Seurat pipeline (right). FIG. 8D: Expression of genes defining broad T cell subsets (CD4, CD8A, CD3E, TRDV1 (T cell receptor delta variable 1)) and other key functional T cell genes (TCF7 (Transcription Factor 7), TOX (Thymocyte Selection-Associated High Mobility Group Box), GZMB (Granzyme B), PRF1 (Perforin 1)). FIG. 8E: Volcano plot showing significantly differentially expressed genes between CD2KO CART19 and CD2WTCART19 in the bulk T cell population. FIG. 8F: Volcano plot showing significantly differentially expressed genes between CD2KOCART19 and CD2WTCART 19 within the Cytotoxic CD8+ T cell subset. FIG. 8G: Pathway analysis of differentially regulated pathways in the Cytotoxic CD8+T cell cluster, comparing CD2KOCART 19 to CD2WTCART 19.

[0147] FIGs. 9A - 91 show that CD2:CD58 signaling axis influences CAR T-cell response. FIG. 9A: Scatterplot of normalized MAGeCK beta scores representing enriched and depleted sgRNAs utilized in a genome- wide knock-out screen of Brunello library-edited Nalm6 cells and CART19, as previously described (Singh, et al., Cancer Discov. 2020 Apr; 10(4): 552-67). FIG. 9B: Overall survival of 17 patients with diffuse large B-cell lymphoma (DLBCL) enrolled to UPCC13413 (NCT02030834) and stratified based on CD58 expression, as determined by bulk tumor RNA sequencing collected prior to CART19 administration. Low CD58: Lowest 1 / 3 of Attorney Docket No. 046483 -7478WO 1(04048) patients based on CD58 gene expression; High CD58: Upper 2 / 3 of patients based on CD58 gene expression. FIG. 9C: Left: Schematic representation of pre-CART19 biopsies from patients with aggressive B-cell lymphomas (Others: marginal zone lymphoma, grey zone lymphoma, and primary mediastinal B-cell lymphoma). Center: CD58 immunohistochemistry H-scores of B-cell lymphoma biopsies obtained prior to CART 19 treatment. Groups include: Long CR: patients who achieved a complete remission (CR) lasting more than 1 year; Short CR: patients with a CR lasting less than 1 year; and Refractory / Relapsed: patients who experienced disease progression or relapse within 90 days of CART 19 infusion. Right: Duration of complete response in days stratified by tertiles of pre-CART19 biopsy CD58 H-score. FIG. 9D: Left: Schematic representation of the pre- and post-CART19 biopsies analyzed. Center: Change in CD58 H-score {delta H-score) in patients with available paired pre- and post-treatment biopsies. “Relapsed” (R) indicates disease progression following a documented complete response, and “Refractory” (Rf) refers to failure to achieve a complete response. Right: Representative CD58 immunohistochemistry images from a patient with DLBCL who experienced relapse after CART19 treatment, showing staining in the pre-treatment biopsy (inset, top) and the post-relapse biopsy (inset, bottom). FIG. 9E: Validation of CD58 knockout in NALM6 cells by flow cytometry. Histogram overlay showing surface CD58 expression in wild-type and CD58 knockout NALM6 cells, as assessed by flow cytometry. Loss of CD58 expression confirms effective gene disruption using the gRNA sequence listed in Table 1. FIG. 9F: Percent cytotoxicity of CART19 cells against OCI-Lyl8 and Nalm6 cells with (CD58WT) and without (CD58KO) CD58 expression across a range of effector-to-target cell ratios after 48 h coculture. Groups compared using ANOVA with Tukey post-hoc multiple comparisons test relative to CD58W 1for each cell type; ** p < 0.01, * p < 0.05. FIG. 9G: Quantification of UTD or CART19 binding avidity to CD58WTand CD58KONalm6 after 15 minutes co-culture on the LUMICKS z- Movi Cell Avidity Analyzer. Upon application of a 1000 pN acoustic force, CAR19 T cells exhibited greater avidity toward NALM6 CD58WTcells compared to NALM6 CD58KOcells, as determined by Student’s t-test (p < 0.05). FIG. 9H: In vivo schema to evaluate the influence of CD58 on CART 19 response. Mice were co-engrafted on day -7 with a 1 : 1 mixture of NALM6 CD58WTand CD58KOcells engineered to express GFP and luciferase. On day 0, mice received 1 x 106CD2WTUTD or CD2WTCART19 cells. Mice were euthanized on day 9 to assess the composition of CD58WTand CD58KOtumor cells in the bone marrow by flow cytometry. FIG. Attorney Docket No. 046483 -7478WO 1(04048)

[0148] 91: Left: Fraction of CD58KONALM6 cells in the bone marrow at day -7 and day 9 in mice treated with CD2WTUTD or CD2WTCART19, as assessed by flow cytometry. Right. Change in the fraction of CD58KOcells between day -7 and day 9. Statistical significance was determined using Student’s t-test; **p < 0.01.

[0149] FIGs. 10A - 10K relate to functional and in vivo validation of the PD-1 :CD2 switch receptor in CART19 cells. FIG. 10A: Schema of a PD-1 :CD2 switch receptor in CD2KOCART19. FIG. 10B: Representative flow cytometry immunophenotyping on day 16 of T-cell expansion of CD2KOCART19 and CD2KOPD-1 :CD2 CART19; y-axis: PD-1-BV421 (representing the extracellular domain of the PD-1 :CD2 switch receptor) and x-axis: G4S-PE (CAR19). FIG. 10C: PD-1 expression over the course of T-cell expansion, representing either the full PD-1:CD2 switch receptor, a truncated version lacking the intracellular CD2 domain, or endogenous physiological PD-1. FIG. 10D: Immunoblot of p-Lck protein in CD2WTCART19, CD2KOCART 19, and CD2KOCART 19 expressing truncated or full PD-1:CD2 switch receptors following 20 minutes of stimulation on plates simultaneously coated with CD 19, PD-L1, and CD58. Bands were derived from the same gel; non-contiguous lanes were juxtaposed, and black lines indicate where the gel was cut. No other modifications were made. FIG. 10E: Densitometry quantification of p-Lck normalized to -actin from the protein lysates shown in FIG. 10D. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post- hoc multiple comparisons test. **p < 0.01, *p < 0.05. FIG. 10F: In vivo experiment where NSG mice were intravenously injected with 1.0 x 106PD-L1 -overexpressing CD58WTNalm6 and subsequently treated with 1.0 x 106CD2KOUTD (n=4), CD2KOCART 19 (n=5), or CD2KOCART19 expressing the PD-1 :CD2 switch receptor (n=6) cells 7 days after leukemia injection (day 0). Total BLI of individual mice treated (dashed lines). Solid lines represent the median of each cohort. Groups were compared using ANOVA with Tukey post-hoc multiple comparisons at each time point measurement, **p < 0.01, * p < 0.05. FIG. 10G: In vivo experiment where NSG mice were intravenously injected with 1.0 x 106PD-L1 -overexpressing CD58KONalm6 and subsequently treated with 1.0 x 106CD2WTUTD (n=4), CD2WTCART19 (n=5), or CD2WTCART19 expressing the PD-1 :CD2 switch receptor (n=6) cells 7 days after leukemia injection (day 0). Total BLI of individual mice treated (dashed lines). Solid lines represent the median of each cohort. Groups were compared using ANOVA with Tukey post-hoc multiple comparisons at each time point measurement. FIG. 10H: Schematic of the A20 lymphoma model (see Attorney Docket No. 046483 -7478WO 1(04048)

[0150] Methods). On day 0, 1 x 10sCD2WTUTD, CD2WTmuCAR19, CD2KOmuCAR19, or CD2KOmuCAR19 expressing the muPD-l:CD2 switch receptor were infused intravenously. FIG. 101: A20 tumor growth curves showing individual mouse replicates following infusion of murine UTD or CART19 T cells. Left. CD2WTUTD and CD2KOmuCAR19. Right. CD2WTmuCAR19 and CD2KOmuCAR19 expressing the muPD-l :CD2 switch receptor. The vertical dashed line indicates the Day 11 measurement. Tumor volume was calculated using the formula: (length x width2) / 2. FIG. 10 J: Tumor volume at Day 11 post-infusion. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post-hoc multiple comparisons test. FIG. 10K: overall survival curves, Wilcox rank-sum test was performed for all survival curve comparisons between CD2KOmuCART19 vs CD2WTmuCART19, CD2KOmuCART19 vs CD2KOmuCART19 expressing the muPD-l :CD2 switch receptor, and CD2WTmuCART19 vs CD2KOmuCART19 expressing the muPD-1 :CD2 switch receptor. * p < 0.05.

[0151] FIGs. 11A - 11H show that CD2 loss alters CART19 activity. FIG. 11A: Population doublings of two, independent, healthy T-cell donor expansions of CART19 with or without CD2 and either a 4-lBBz (CART19-BBz) or CD28z (CART19-28z) costimulatory domain. FIG. 11B: Cytotoxicity assay after 72 hours of co-culture between CD2WTUTD, CD2KOUTD, CD2WTCART19, or CD2KOCART19 T cells and luciferase-expressing NALM6 target cells. Cytotoxic activity was measured by luminescence reduction, which reflects viable tumor cell content. Effector-to-target (E:T) ratios tested were 0.25: 1, 0.125: 1, and 0.0625: 1. FIG. 11C: Proliferation assay of CD2WTUTD, CD2KOUTD, CD2WTCAR19, and CD2KOCAR19 T cells co-cultured with irradiated NALM6 cells (E: T ratio of 0.25: 1). T cells were labeled with CellTrace Violet prior to co-culture and analyzed by flow cytometry after 5 days. The graph shows the fold change in the absolute number of CD3+T cells compared to baseline (day 0), measured using counting beads. FIG. 11D: Metabolic profiling of CD2WTUTD, CD2WTCAR19, and CD2KOCAR19 T cells using a Seahorse XF assay. 3xl06CAR+ T cells (or equivalent numbers of UTD) were cultured for 48 hours on plates coated simultaneously with recombinant CD 19 and CD58 proteins prior to analysis (ns, Ordinary one-way ANOVA). FIG. 11E: Replicative bioluminescence imaging of leukemia burden engrafted with 1.0 x 106Nalm6 (left) and overall survival (right) of composite NSG mice treated with 1.0 x 106Mock UTD, CD2KOUTD, CD2- wild type (CD2WT) CART19-BBz, and CD2KOCART19-BBz cells from a separate, independent healthy T-cell donor. n= 3-6 mice per group. FIG. HF: Body mass of each NSG mouse in both Attorney Docket No. 046483 -7478WO 1(04048) donor cohorts. FIG. 11G: Serum cytokine quantification of NSG mouse plasma fractionated from peripheral blood at Day 7 post CD2WTUTD, CD2KOUTD, CD2WTCART 19, and CD2KOCART19 treatment, as determined by Luminex multiplex assay. FIG. 11H: Left: Bioluminescence imaging (BLI) of leukemia burden in NSG mice engrafted with 1.0 x 106NALM6 cells and treated with 1.0 x io6T cells: CD2WTUTD, CD2KOUTD, CD2WTCART19- 28z, or CD2KOCART19-28Z. Dashed lines represent total BLI signal from individual mice over time; solid lines indicate the median signal per group. Statistical analysis was performed by oneway ANOVA followed by Tukey’s post-hoc multiple comparisons test. Right: Overall survival of NSG mice from the experiment shown at left. Wilcoxon rank-sum test was used to compare CD2WTCART19 and CD2KOCART19 groups; **p < 0.01, *p < 0.05.

[0152] FIGs. 12A - 12D relate to Ancillary single-cell RNA sequencing analyses of in vivo- derived CD2KOCART19 cells. FIG. 12A: Immunophenotyping of CD2WTUTD (untreated, left) and CD2WTCART19 (middle) T cells prior to injection into NSG mice, corresponding to FIG. 8A. To ensure complete CD2 deficiency for single-cell analyses, CD2KOCART19 cells were additionally FACS-sorted based on CD2 expression prior to infusion (right). FIG. 12B: Schematic of the workflow on day 16 post-infusion for mouse-specific barcoding. Following PBMC isolation, red blood cells were lysed, and cells from each mouse were individually stained for T cells (CD2 / CD3 / CD5-APC) and uniquely tagged with a barcoding antibody (TotalSeq-A) targeting CD45 and MHC Class I. CD2WTand CD2KOT cell populations were then loaded as separate samples onto the 10X Chromium Controller for single-cell RNA sequencing. FIG. 12C: UMAP of the distribution of TotalSeq-A barcodes identifying individual mice in the Mock CART19 (n = 8) and CD2KOCART19 (n = 6) groups. FIG. 12D: Single cell expression levels of key genes involved in T cell activation (LCK, l l'l, PLCH1) and exhaustion (LAG3, HAVCR2, PDCD1) left), as well as transcription factors (TBX21, GATA3, RORC, and F0XP3) associated with distinct T cell subset phenotypes (right).

[0153] FIGs. 13A - 13M relate to CD2:CD58 signaling axis rescue strategies. FIG. 13A: Schema of in cis CD2 intracellular rescue in CD2-deficient CART 19. FIG. 13B: In vitro cytotoxicity of CD2KOCART19.28z and CD2KOCART 19.28. CD2z (CART19-iCD2) 72 hours after coculture with either CD58WTor CD58KOOCI-Lyl8 cells. Percent cytotoxicity for each CART 19 group against CD58KOOCI-Lyl8 was normalized against percent cytotoxicity against CD 8WTOCI-Lyl8. Two-way ANOVA with Tukey multiple comparison post-hoc test was used Attorney Docket No. 046483 -7478WO 1(04048) to determine statistical significance ** p < 0.01 , * p < 0.05. FIG. 13C: NSG mice were intravenously injected with 1.0 x 106CD58WTNalm6 and subsequently treated with 1.0 x 106untransduced T-cells (CD2WTUTD, n=3), 1.0 x 106CAR-positive CD2KOCART19.28z (n=6), or 1.0 x 106CAR-positive CD2KOCART19-iCD2 (n=6) seven days after leukemia injection. Total BLI (left) and overall survival (right) is shown; ns = not significant. FIG. 13D: Construct maps of a PD-1 :CD2 intracellular signaling domain switch receptor integrated with a P2A selfcleaving peptide and the CAR19.BBz construct, and its corresponding truncated version lacking the intracellular CD2 domain. FIG. 13E: Population doublings, as calculated by normalized viable cell count relative to the total number of cells initially electroporated, of each product cohort derived from a single healthy T-cell donor. FIG. 13F: Cytotoxicity of CD2WTUTD, CD2WTCART 19, CD2WTPD-1 truncated CART 19, and CD2WTPD-1 :CD2 CART 19 against CD58KONALM6 cells with variable surface PDL1 expression. PDL1 -expressing CD58KONALM6 cells were generated by lentiviral transduction with a PD-L1 -overexpressing vector and subsequently mixed at defined ratios with PD-L1 wild-type CD58KONALM6 cells to obtain target populations with approximately 0%, 20%, 45%, and 100% PDL1+cells. FIG. 13G: Schematic representation of the Jurkat triple reporter cell line, engineered to report T-cell activation through NFAT-GFP, NF-KB-mCherry, and AP-l-CFP fluorescent readouts. FIG. 13H: Fold change in MFI of NFAT-GFP, NF-KB-mCherry, and AP-l-CFP in Jurkat triple reporter cells after 24 hours of co-culture with CD58WTNALM6 cells. Conditions include CD2WTUTD, CD2WTCART 19, CD2WTCART 19 expressing the truncated switch receptor, and CD2W TCART19 expressing the full PD-1 :CD2 switch receptor (E:T 1 : 1). FIG. 131: Fold change in MFI of NFAT-GFP, NF-KB-mCherry, and AP-l-CFP in Jurkat triple reporter cells after 24 hours of co-culture with CD58KONALM6 overexpressing PD-L1. Conditions include CD2W 1UTD, CD2WTCART 19, CD2WTCART 19 expressing the truncated switch receptor, and CD2WTCART 19 expressing the full PD-1 :CD2 switch receptor (E:T 1 :1). FIG. 13J: Overall survival curves relative to FIG. 10F, Wilcox rank-sum test was performed for all survival curve comparisons between CD2KOCART19 and CD2KOCART19 expressing the PD-ECD2 switch receptor (p=0.001). FIG. 13K: Overall survival curves relative to FIG. 10G, Wilcox rank-sum test was performed for all survival curve comparison between CD2WTCART19 and CD2WTCART19 expressing the PD-1 :CD2 switch receptor (p=0.0316). FIG. 13L: Knockout efficiency of murine CD2 measured by flow cytometry following gRNA screening. FIG. 13M: Expression Attorney Docket No. 046483 -7478WO 1(04048) of the murine switch receptor (murineCAR19_T2A_murine_PD-l :CD2) assessed by flow cytometry in the murine CD2KOT-cell population at Day 8 of murine T-cell expansion.

[0154] DETAILED DESCRIPTION The present invention provides compositions and methods to enhance the anti-cancer efficacy of adoptive cell transfer immunotherapies (e.g., CAR T cell therapy).

[0155] In one aspect, the invention provides a chimeric switch receptor comprising (i) an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a CD2 intracellular signaling domain.

[0156] In another aspect, the invention provides a nucleic acid comprising (i) a first nucleotide sequence encoding a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and (ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

[0157] In another aspect, the invention provides a modified immune cell or precursor cell thereof, comprising (i) a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and (ii) a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

[0158] In another aspect, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of modified immune cells or precursor cells thereof comprising (i) a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and (ii) a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR). Attorney Docket No. 046483 -7478WO 1(04048)

[0159] In other aspects, provided herein are related compositions (e.g., pharmaceutical compositions) and kits.

[0160] It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0161] Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0162] Methods and techniques using T Cells with chimeric antigen receptors (CAR T cells) are described in e.g., Ruella, et al., J. Clin. Invest., 126(10):3814-3826 (2016) and Kalos, et al., 3 (95), 95ra73 : 1-11 (2011), the contents of which are hereby incorporated by reference in their entireties.

[0163] Definitions

[0164] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0165] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited Attorney Docket No. 046483 -7478WO 1(04048) and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0166] That the disclosure may be more readily understood, select terms are defined below.

[0167] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0168] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0169] “Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0170] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0171] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

[0172] Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily Attorney Docket No. 046483 -7478WO 1(04048) apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0173] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0174] A “co- stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

[0175] A “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0176] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0177] The term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes.

[0178] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies Attorney Docket No. 046483 -7478WO 1(04048) and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0179] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0180] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and / or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and / or sugars in size. Preferably, the epitope is about 4- 18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0181] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system. For example, a chimeric antigen receptor can be produced in a cell by the introduction of an exogenous nucleic acid molecule encoding the chimeric antigen receptor. A nucleic acid molecule that is introduced into the cell can also be referred to as a “heterologous” nucleic acid molecule. The protein produced by an exogenous nucleic acid molecule can also be referred to as a “heterologous protein”.

[0182] The term “expand” as used herein refers to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number Attorney Docket No. 046483 -7478WO 1(04048) relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term “ex vivo ” as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0183] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0184] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0185] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0186] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0187] The term “immunosuppressive” is used herein to refer to reducing overall immune response.

[0188] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or Attorney Docket No. 046483 -7478WO 1(04048) peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0189] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0190] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0191] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0192] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0193] The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T ”

[0194] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). Attorney Docket No. 046483 -7478WO 1(04048)

[0195] “Parenteral” administration of an immunogenic composition includes, e g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0196] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, “nucleic acid” and “polynucleotide” as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides” and which comprise one or more “nucleotide sequence(s)”. The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences (i.e., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0197] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0198] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. Attorney Docket No. 046483 -7478WO 1(04048)

[0199] However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0200] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and / or reorganization of cytoskeletal structures, and the like.

[0201] A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

[0202] A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

[0203] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used herein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as simian and non-human primate mammals. Preferably, the subject is human.

[0204] A “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic Attorney Docket No. 046483 -7478WO 1(04048) nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0205] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (P) chain, although in some cells the TCR consists of gamma and delta (y / 5) chains. TCRs may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell. In some embodiments, the cells can comprises a heterologous TCR. TCRs can be modified to bind to a specific antigen or based on a TCR from a subject and placed in a different cell than it was originally isolated from. Examples of TCRs that can be heterologously expressed in a cell include those that are described and provided for in U.S. Patent Application Publication No. 20190359678, PCT Publication No. W02020038491, U.S. Patent Application Publication No. 20210355189A1, U.S. Patent No. 8,519,100, U.S. Patent No. 8,216,565, U.S. Patent No. 9,688,739, U.S. Patent Application Publication No. 20110189141, U.S. Patent Application Publication No. 20220168346, U.S. Patent Application Publication No. 20220168347, U.S. Patent Application Publication No. 20170015727, U.S. Patent Application Publication No. 20200054678, U.S. Patent No. 10,117,918, U.S. Patent No. 11,471,489, U.S. Patent No. 10,464,987, U.S. Patent No. 11,686,724, each of which is hereby incorporated by reference in its entirety. In some embodiments, the TCR is a TCR that binds to gplOO, a TCR that binds to a tumor antigen or MHC presented tumor antigen, other non-naturally occurring TCRs, and the like.

[0206] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0207] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, Attorney Docket No. 046483 -7478WO 1(04048) transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0208] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0209] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0210] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0211] CD2 Chimeric Switch Receptors and Modified Immune Cells

[0212] It is established that CD2 has a costimulatory role and that CD58, the ligand of CD2, is downregulated in certain cancers. As demonstrated herein, lack of CD2 attenuates CAR T cell efficacy by reducing avidity for tumor antigen, costimulation, and ultimately in vivo activity. Analogously, it is demonstrated herein that tumor CD58 loss reduces CAR T cell efficacy in the clinical setting. As such, it is contemplated herein that a CD2 chimeric switch receptor (e.g., a PD-1 :CD2 chimeric switch receptor) will rescue CD2:CD58 signaling in modified immune cells (e g., CAR T cells), thereby increasing immunotherapy efficacy. Attorney Docket No. 046483 -7478WO 1(04048)

[0213] As demonstrated herein in Example 1, an exemplary CD2 chimeric switch receptor comprising a CD2 intracellular signaling domain linked to a PD-1 ligand binding domain (PD- 1 :CD2) expressed on CAR T cells effectively rescued intracellular CD2 signaling in the absence of CD2:CD58 signaling, resulting in improved in vivo outcomes. This exemplary PD-LCD2 switch receptor recovered in vivo CAR T cell efficacy as compared to intracellular CD2 insertion in cis with anti-CD19 CAR T cells. Without wishing to be bound by theory, one hypothesis for this effect is that the proximal positioning of the CD2 intracellular domain to the cell surface might enhance CD2-mediated costimulation upon PD-1-PD-L1 engagement.

[0214] In one aspect, the invention provides a chimeric switch receptor comprising (i) an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a CD2 intracellular signaling domain.

[0215] In certain embodiments, the chimeric switch receptor comprises (i) an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a CD2 intracellular signaling domain. In some embodiments, the inhibitory immunoreceptor is Programmed Cell Death Protein 1 (PD-1). In some embodiments, the chimeric switch receptor comprises (i) a PD- 1 LBD, (ii) a transmembrane domain, and (iii) an intracellular domain comprising a CD2 intracellular signaling domain.

[0216] In some embodiments, the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T- lymphocyte- Associated Protein 4 (CTLA4).

[0217] In certain embodiments, the chimeric switch receptor comprises (i) an extracellular domain comprising an anti-checkpoint inhibitor antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a CD2 intracellular signaling domain. In some embodiments, the checkpoint inhibitor is Programmed Cell Death Ligand-1 (PD-L1). In some embodiments, the chimeric switch receptor comprises (i) an anti-PD-Ll antigen binding Attorney Docket No. 046483 -7478WO 1(04048) domain, (ii) a transmembrane domain, and (iii) an intracellular domain comprising a CD2 intracellular signaling domain.

[0218] In some embodiments, the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand- 1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte-Associated Protein 4 (CTLA4).

[0219] In some embodiments, the chimeric switch receptor comprises a CD2 transmembrane domain.

[0220] In certain embodiments, the chimeric switch receptor comprises a PD-1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

[0221] In certain embodiments, the chimeric switch receptor comprises an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

[0222] In some embodiments, the anti-checkpoint inhibitor antigen binding domain comprises a single-chain variable fragment (scFv).

[0223] In certain embodiments, the chimeric switch receptor comprises an anti-PD-Ll scFv, a CD2 transmembrane domain, and a CD2 intracellular signaling domain

[0224] It will be understood by those of skill in the art that the chimeric switch receptor may comprise the indicated domains from proteins (e.g., PD-1, CD2) derived from a human or nonhuman mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as simian and non-human primate mammals. In certain embodiments, the proteins from which the chimeric switch receptor is derived are human proteins. In certain embodiments, the proteins from which the chimeric switch receptor is derived are non-human mammalian proteins. In some embodiments, the proteins from which the chimeric switch receptor is derived are selected from the group consisting of non-human primate proteins, simian proteins, ovine proteins, bovine proteins, porcine proteins, canine proteins, feline proteins, and murine proteins.

[0225] In certain embodiments, the chimeric switch receptor comprises (i) an extracellular domain comprising a ligand binding domain (LBD) of a human inhibitory immunoreceptor (e.g., a human PD-1 LBD) or an antigen binding domain that targets a human checkpoint inhibitor (e.g., an anti -human PD-L1 antigen binding domain; (ii) a human protein transmembrane domain Attorney Docket No. 046483 -7478WO 1(04048)

[0226] (e g., a human CD2 transmembrane domain); and (iii) an intracellular domain comprising a human CD2 intracellular signaling domain.

[0227] In certain embodiments, the chimeric switch receptor comprises the following amino acid sequence: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSN TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN D SGT YLCGAISL APK AQIKE SLRAELRVTERRAE VPT AHP SP SPRP AGQFQTLVIYLIIGIC GGGSLLMVFVALLVFYITKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNP ATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPK PPHGAAENSLSPSSN (SEQ ID NO: 1).

[0228] In some embodiments, the chimeric switch receptor comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 1.

[0229] In certain embodiments, the chimeric switch receptor comprises a leader sequence. In some embodiments, the leader sequence comprises the following amino acid sequence: MQIPQAPWPVVWAVLQLGWRPGW (SEQ ID NO: 2). In some embodiments, the leader sequence comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 2.

[0230] In certain embodiments, the chimeric switch receptor comprises a PD-1 LBD. In some embodiments, the PD-1 LBD comprises the following amino acid sequence:

[0231] FLD SPDRPWNPPTF SP ALL VVTEGDN ATFTC SF SNT SESF VLNWYRMSP SNQTDKL AAFP EDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAEL RVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 3). In some embodiments, the PD-1 LBD comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 3. Attorney Docket No. 046483 -7478WO 1(04048)

[0232] In certain embodiments, the chimeric switch receptor comprises a CD2 transmembrane domain. In some embodiments, the CD2 transmembrane domain comprises the following amino acid sequence: IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO: 4). In some embodiments, the CD2 transmembrane domain comprises an amino acid sequence having at least 80%, at least

[0233] 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least

[0234] 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

[0235] 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID

[0236] NO: 4.

[0237] In certain embodiments, the chimeric switch receptor comprises a CD2 intracellular signaling domain. In some embodiments, the CD2 intracellular signaling domain comprises the following amino acid sequence: KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSH RPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 5). In some embodiments, the CD2 intracellular signaling domain comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 5.

[0238] In another aspect, the invention provides a modified immune cell or precursor cell thereof, comprising a chimeric switch receptor of the invention and a chimeric antigen receptor (CAR) or a heterologous TCR. In some embodiments, the modified immune cell or precursor cell thereof (e.g., T cell) comprises (i) a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and (ii) a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

[0239] Chimeric switch receptors of the invention are described herein. Exemplary suitable CARs and TCRs are known in the art and are described herein. In some embodiments, the CAR or the TCR targets a tumor antigen. The modified immune cells can comprise any CAR and / or any heterologous TCR disclosed herein or known in the art. Attorney Docket No. 046483 -7478WO 1(04048)

[0240] Various approaches and techniques for obtaining and / or modifying an immune cell, or precursor cell thereof, comprising two or more heterologous / exogenous proteins are known in the art. In some embodiments, the modified immune cell or precursor cell thereof comprises a nucleic acid disclosed herein. In certain embodiments, the modified immune cell or precursor cell thereof comprises a nucleic acid comprising (i) a first nucleotide sequence encoding a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anticheckpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and (ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

[0241] In certain embodiments, the modified immune cell or precursor cell thereof comprises a vector (e.g., an expression vector) comprising a nucleic acid disclosed herein. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a retroviral vector.

[0242] In certain embodiments, the modified immune cell is an autologous cell in reference to the subject that the cell is eventually administered to. In certain embodiments, the modified immune cell is a cell isolated from a human subject. Suitable exemplary immune cells for use in the invention include, but are not limited to, a T cell, a natural killer (NK) cell, and a macrophage. In some embodiments, the T cell is a CD8+ T cell, a natural killer T (NKT) cell, or a gamma-delta (yS) T cell.

[0243] In certain embodiments, the modified immune cell is a human cell. In certain embodiments, the modified immune cell is an autologous cell. In certain embodiments, the modified immune cell is an autologous cell obtained from a human. In certain embodiments, the modified immune cell is a T cell. In certain embodiments, the modified immune cell is a human T cell. In certain embodiments, the modified immune cell is an autologous human T cell.

[0244] In some aspects, the provided compositions and methods include those in which at least or greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of immune cells in a composition or population of immune cells comprise both the chimeric switch receptor of the invention and a CAR or a heterologous TCR. In some embodiments, the provided compositions and methods include those in which at least or greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of immune cells in a composition or population of immune cells Attorney Docket No. 046483 -7478WO 1(04048) comprise a vector (e.g., a lentiviral vector) which expresses both the chimeric switch receptor described herein and a CAR or a heterologous TCR.

[0245] Chimeric Antigen Receptors

[0246] In some embodiments, the CAR is a recombinant CAR. In some embodiments, the CAR is encoded by a heterologous sequence (e.g., transgene) present in the immune cell.

[0247] In certain embodiments, the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain. The antigen binding domain of the CAR is operably linked to another domain of the CAR, such as a hinge, a transmembrane domain or an intracellular domain, each described elsewhere herein, for expression in the cell. In one embodiment, a first nucleotide sequence encoding the antigen binding domain is operably linked to a second nucleotide sequence encoding a hinge and / or transmembrane domain, and further operably linked to a third nucleotide sequence encoding an intracellular domain.

[0248] The antigen binding domain described herein can be combined with any of the transmembrane domains described herein, any of the intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in a CAR of the present invention, such as a hinge domain or a spacer sequence.

[0249] The CAR of the present invention may also include a leader sequence as described herein. The CAR of the present invention may also include a hinge domain as described herein. The CAR of the present invention may also include one or more spacer domains or linkers as described herein which may serve to link one domain of the CAR to the next domain.

[0250] Antigen Binding Domain

[0251] The antigen binding domain of a CAR is an extracellular region of the CAR for binding to a specific target antigen including proteins, carbohydrates, and glycolipids. The CAR of the invention comprises an antigen binding domain that is capable of binding a tumor antigen. Suitable tumor antigens are known in the art and include, but are not limited to, alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein Attorney Docket No. 046483 -7478WO 1(04048)

[0252] (FBP), GD-2, Glycolipid F77, glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, VEGFR2, and any combination thereof. In some embodiments, the tumor antigen is CD19. In some embodiments, the antigen-binding domain is an anti-CD19 antigen binding domain which is capable of binding CD 19, such as the FMC63 scFv known in the art.

[0253] The antigen binding domain can include any domain that binds to the antigen (e.g., tumor antigen) and may include, but is not limited to, a monoclonal antibody (mAb), a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-domain antibody, a full-length antibody or any antigen-binding fragment thereof, a Fab, and a single-chain variable fragment (scFv). In some embodiments, the antigen binding domain comprises an aglycosylated antibody or a fragment thereof or scFv thereof.

[0254] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The variable heavy (VH) and light (VL) chains are either joined directly or joined by a peptide linker, which connects the N- terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N- terminus of the VL. In some embodiments, the antigen binding domain (e.g., tumor antigen binding domain) comprises an scFv having the configuration from N-terminus to C-terminus, VH - linker - VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL - linker - VH or VH - linker -VL. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

[0255] The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention. In one Attorney Docket No. 046483 -7478WO 1(04048) embodiment, an antigen binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are separated by a linker sequence.

[0256] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3): 173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71 ; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0257] As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0258] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab’) (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0259] In other embodiments, the antigen binding domain comprises an antibody mimetic protein such as, for example, designed ankyrin repeat protein (DARPin), affibody, monobody, (i.e., adnectin), affilin, affimer, affitin, alphabody, avimer, Kunitz domain peptide, or anticalin. Constructs with specific binding affinities can be generated using DARPin libraries e.g., as described in Seeger, et al., , Protein Sci., 22:1239-1257 (2013). Attorney Docket No. 046483 -7478WO 1(04048)

[0260] In some embodiments, the antigen binding domain may be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a human antibody or a fragment thereof. In some embodiments, the antigen binding domain may be derived from a different species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a murine antibody or a fragment thereof, or a humanized murine antibody or a fragment thereof.

[0261] 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 regions (LCDRs). In certain embodiments, the antigen binding domain comprises a linker.

[0262] Transmembrane Domain

[0263] CARs of the present invention may comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain of the CAR. The transmembrane domain of the CAR is a region that is capable of spanning the plasma membrane of a cell ( .g., an immune cell or precursor thereof). In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR.

[0264] In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex.

[0265] The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane domain of particular use in this invention include, without limitation, transmembrane domains 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, CD7, CD8, CD9, CD16, CD22, Attorney Docket No. 046483 -7478WO 1(04048)

[0266] CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), ICOS, CD278, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

[0267] In certain embodiments, the transmembrane domain comprises a transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is a transmembrane domain of CD8a.

[0268] In some embodiments, 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, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0269] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in the CAR.

[0270] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also include a hinge region. The hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4).

[0271] In some embodiments, the CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. The hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell Attorney Docket No. 046483 -7478WO 1(04048)

[0272] (Hudecek et al., supra . The flexibility of the hinge region permits the hinge region to adopt many different conformations.

[0273] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0274] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more.

[0275] Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

[0276] For example, hinge regions include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). The hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region (see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897). In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof. Attorney Docket No. 046483 -7478WO 1(04048)

[0277] Intracellular Signaling Domain

[0278] The CAR of the present invention also includes an intracellular signaling domain. The terms “intracellular signaling domain” and “intracellular domain” are used interchangeably herein. The intracellular signaling domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., immune cell). The intracellular signaling domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and / or destroying a target cell.

[0279] Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co- stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.

[0280] Examples of the intracellular signaling domain include, without limitation, the C, chain of the T cell receptor complex or any of its homologs, e.g., q chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 8 and 8), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may comprise an intracellular signaling domain of a protein selected from human CD3 zeta chain, FcyRIII, FcsRI, DAP10, DAP12, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptors, and combinations thereof.

[0281] In one embodiment, the intracellular signaling domain of the CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, such as any synthetic sequence thereof, that has the same functional capability, and any combination thereof.

[0282] Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP 10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4- IBB (CD 137), OX9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, Attorney Docket No. 046483 -7478WO 1(04048)

[0283] CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co- stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co- stimulatory molecule that has the same functional capability, and any combination thereof.

[0284] Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NK.T cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g, Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0285] Intracellular signaling domains suitable for use in the CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) IT AM motifs as described below. In some embodiments, the intracellular signaling domain includes DAP10 / CD28 type signaling chains. In some embodiments, the Attorney Docket No. 046483 -7478WO 1(04048) intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.

[0286] Intracellular signaling domains suitable for use in the CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, an ITAM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of the CAR comprises 3 ITAM motifs.

[0287] In some embodiments, intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine based activation motifs (IT AMs) such as, but not limited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0288] A suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0289] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell Attorney Docket No. 046483 -7478WO 1(04048) surface glycoprotein CD3 delta chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T- cell surface antigen T3 / Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig- alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In one embodiment, an intracellular signaling domain suitable for use in a CAR of the present disclosure includes a DAP10 / CD28 type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in a CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 zeta.

[0290] While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire molecule. To the extent that a truncated portion of the intracellular signaling 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 intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0291] The intracellular domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR.

[0292] In certain embodiments, the intracellular domain comprises a costimulatory domain of 4- 1BB. In certain embodiments, the intracellular domain comprises an intracellular domain of Attorney Docket No. 046483 -7478WO 1(04048)

[0293] CD3(^ or a variant thereof. In certain embodiments, the intracellular domain comprises a costimulatory domain of 4- IBB and an intracellular domain of CD3(^.

[0294] Tolerable variations of the individual CAR domain sequences (leader, antigen binding domain, hinge, transmembrane, and / or intracellular domains) will be known to those of skill in the art. For example, in certain embodiments the CAR domain comprises an amino acid sequence that has 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 naturally-occurring or known sequence.

[0295] In one aspect, the invention provides a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the CAR comprises an anti-CD19 antigen binding domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular domain.

[0296] In some embodiments, the CAR is the CTL019 CAR comprising the following amino acid sequence: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSW IRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCA KHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

[0297] HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR (SEQ ID NO: 6).

[0298] In some embodiments, the CAR is the CTL019 CAR encoded by the following nucleotide sequence: atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc Attorney Docket No. 046483 -7478WO 1(04048) ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgc ( SEQ ID NO : 7 ) .

[0299] T Cell

[0300] In some embodiments, the TCR is a heterologous or exogenous TCR. In some embodiments, the TCR is encoded by a heterologous sequence (e.g., transgene) present in the immune cell.

[0301] TCRs can be modified to bind to a specific antigen or based on a TCR from a subject and placed in a different cell than it was originally isolated from. Examples of TCRs that can be heterologously expressed in a cell include those that are described and provided for in U.S. Patent Application Publication No. 20190359678, PCT Publication No. W02020038491, U.S. Patent Application Publication No. 20210355189A1, U.S. Patent No. 8,519,100, U.S. Patent No. 8,216,565, U.S. Patent No. 9,688,739, U.S. Patent Application Publication No. 20110189141, U.S. Patent Application Publication No. 20220168346, U.S. Patent Application Publication No. 20220168347, U.S. Patent Application Publication No. 20170015727, U.S. Patent Application Publication No. 20200054678, U.S. Patent No. 10,117,918, U.S. Patent No. 11,471,489, U.S. Patent No. 10,464,987, U.S. Patent No. 11,686,724, each of which is hereby incorporated by reference in its entirety. In some embodiments, the TCR is a TCR that binds to gplOO, a TCR that binds to a tumor antigen or MHC presented tumor antigen, other non-naturally occurring TCRs, and the like. Attorney Docket No. 046483 -7478WO 1(04048)

[0302] In some embodiments, the TCR targets (i.e., has antigenic specificity for) an antigen, for example, a tumor antigen. As used herein, the phrase “having antigenic specificity,” or like phrase, means that the TCR can specifically bind to and recognize the antigen, or an epitope thereof.

[0303] Natural TCRs are generally heterodimers. In humans, in 95% of T cells, the TCR comprises an alpha (a) chain and a beta (0) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells, the TCR comprises gamma and delta (y / 8) chains (encoded by TRG and TRD, respectively). Natural TCR complexes are an octameric assembly of type-I singlespanning membrane proteins arranged into four dimeric modules: the variable ligand-binding TCRaP module (in most T cells) (or the TCR y / 8 module) and the three invariant signaling modules CD38s, CD3ys, and CD3^ dimer. The TCR.c / 0 module binds to pMHC ligands on APC or target cell surfaces, but these proteins lack intrinsic signaling capability and, as such, rely on the signaling modules to transmit information through their cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). See, e.g., Chandler et al. IntJMol Set (2020) 21 :7424, which is incorporated by reference herein. Natural TCRs can be cloned and modified using standard molecular biology and genetic engineering techniques known in the art.

[0304] Various engineered TCR forms are also known in the art, including TCR mimic antibodies (Chang et al., Expert Opin Biol Ther. (2016) 16(8):979-87), TCR-like CARs and TCR-CARs (Walseng et al., Sci Rep. (2017) 7: 1-10; Akatsuka et al., Front Immunol. (2020) 11 :257; Poorebrahim et al., Cancer Gene Ther. (2021) 28(6): 581 -589. Unlike CARs, TCRs are not restricted to the cell surface antigens, but can detect and bind to the peptides presented by MHC molecules (pMHCs). This feature provides a wide range of potential targets for TCRs such as tumor-specific neoepitopes. Of note, redirection of TCR-based CARs on the highly tumorspecific neoepitopes can prevent “off-tumor” toxicities that are commonly associated with CAR therapies.

[0305] In some embodiments, the TCR is a natural TCR. In some embodiments, the TCR is a modified TCR. In some embodiments, the TCR is an engineered TCR, such as a TCR mimic or antibody-like structure, a CAR-like TCR, or a CAR-TCR. In some embodiments, the TCR is a murine TCR. In some embodiments, the TCR is a human TCR. In some embodiments, the TCR is a hybrid TCR having one or more portions of a human TCR (e.g., a constant portion or a variable portion) and one or more portions of a murine TCR (e.g., a constant portion or a variable Attorney Docket No. 046483 -7478WO 1(04048) portion). Alternatively, the portion can be a few amino acids of a human TCR, such that the TCR, which is mostly murine, is “humanized.” Methods of making such hybrid TCRs are known in the art (see, for example, Cohen et al., Cancer Res., (2006) 66:8878-8886).

[0306] In some embodiments, the TCR targets a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7- H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD 19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), gplOO, HER2, HLA-A2, HPV E6, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MART- 1, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D- Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PRAME, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

[0307] In some embodiments, the TCR targets (z.e., has antigenic specificity for) a gplOO melanoma antigen, e.g., human gplOO. In some embodiments, the tumor antigen is gplOO. gplOO, also known in the art as SILV, SI, SIL, ME20, PMEL17, or D12S53E. gplOO, is a protein known to play an important role in regulating mammalian pigmentation (Hoashi et al., J. Biol. Chem. (2005) 280: 14006-14016) and is known as a cancer antigen expressed by human tumors, including melanoma and colorectal tumors (Tartaglia et al., Vaccine (2001) 19(17- 19):2571 -5). The amino acid and nucleotide sequences of human gplOO are published in the GenBank database of the National Center for Biotechnology Information (NCBI) as GenBank Accession No. NP_008859 (amino acid sequence) and GenBank Accession No. NM_006928.3 (nucleotide sequence).

[0308] TCRs having antigenic specificity for gplOO (i.e., anti-gplOO TCRs) are known in the art, such as the TCRs described in, e.g., US20140219978A1. In some embodiments, the TCR is a pmel-1 TCR. The pmel-1 mouse model was developed as a system to model treatment of malignant melanoma using adoptive cell therapy (ACT) (Overwijk, et al., J Exp Med. (2003), 198(4): 569-80). The target antigen, pmel-17, is an ortholog of the melanocyte differentiation antigen gplOO, which is often overexpressed in human melanomas. Attorney Docket No. 046483 -7478WO 1(04048)

[0309] In some embodiments, the TCR targets (z.e., has antigenic specificity for) an NY-ESO-1 antigen. NY-ESO-1 is a cancer-testis antigen overexpressed in synovial sarcoma, myxoid liposarcoma, melanoma and other tumors. In some embodiments, the TCR is an NYESO1-TCR clone 1G4 (Robbins, et al., J Immunol, 2008, 180:6116-6131). In some embodiments, the TCR targets an antigen selected from MAGE-A3 / A6, MAGE-

[0310] A10, AFP, PRAME, MART-1, and HPV E6.

[0311] Nucleic Acids and Expression Vectors

[0312] The present disclosure provides certain nucleic acids for encoding proteins of the present invention, e.g., a CAR, a TCR, and / or a chimeric switch receptor of the invention.

[0313] In one aspect, the invention provides a nucleic acid comprising (i) a first nucleotide sequence encoding a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and (ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

[0314] Suitable CARs and TCRs are known in the art. Exemplary CARs and TCRs are described herein. In some embodiments, the CAR or the TCR targets a tumor antigen.

[0315] In some embodiments, the nucleic acid further comprises a self-cleaving peptide sequence between the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the self-cleaving peptide sequence is a P2A sequence.

[0316] In certain embodiments, the encoded chimeric switch receptor comprises (i) an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a CD2 intracellular signaling domain. In some embodiments, the inhibitory immunoreceptor is Programmed Cell Death Protein 1 (PD-1). In some embodiments, the encoded chimeric switch receptor comprises (i) a PD-1 LBD, (ii) a transmembrane domain, and (iii) an intracellular domain comprising a CD2 intracellular signaling domain.

[0317] In some embodiments, the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Attorney Docket No. 046483 -7478WO 1(04048)

[0318] Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T- lymphocyte- Associated Protein 4 (CTLA4).

[0319] In certain embodiments, the encoded chimeric switch receptor comprises (i) an extracellular domain comprising an anti-checkpoint inhibitor antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a CD2 intracellular signaling domain. In some embodiments, the checkpoint inhibitor is Programmed Cell Death Ligand-1 (PD-L1). In some embodiments, the encoded chimeric switch receptor comprises (i) an anti-PD-Ll antigen binding domain, (ii) a transmembrane domain, and (iii) an intracellular domain comprising a CD2 intracellular signaling domain.

[0320] In some embodiments, the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand-1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte-Associated Protein 4 (CTLA4)

[0321] In some embodiments, the encoded chimeric switch receptor comprises a CD2 transmembrane domain.

[0322] In certain embodiments, the encoded chimeric switch receptor comprises a PD-1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

[0323] In certain embodiments, the chimeric switch receptor comprises a PD-1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

[0324] In certain embodiments, the chimeric switch receptor comprises an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

[0325] In some embodiments, the anti-checkpoint inhibitor antigen binding domain comprises a single-chain variable fragment (scFv).

[0326] In certain embodiments, the chimeric switch receptor comprises an anti-PD-Ll scFv, a CD2 transmembrane domain, and a CD2 intracellular signaling domain

[0327] It will be understood by those of skill in the art that the chimeric switch receptor may comprise the indicated domains from proteins (e.g., PD-1, CD2) derived from a human or nonhuman mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as simian and non-human primate Attorney Docket No. 046483 -7478WO 1(04048) mammals. In certain embodiments, the proteins from which the chimeric switch receptor is derived are human proteins. In certain embodiments, the proteins from which the chimeric switch receptor is derived are non-human mammalian proteins. In some embodiments, the proteins from which the chimeric switch receptor is derived are selected from the group consisting of non-human primate proteins, simian proteins, ovine proteins, bovine proteins, porcine proteins, canine proteins, feline proteins, and murine proteins.

[0328] In certain embodiments, the chimeric switch receptor comprises (i) an extracellular domain comprising a ligand binding domain (LBD) of a human inhibitory immunoreceptor (e.g., a human PD-1 LBD) or an antigen binding domain that targets a human checkpoint inhibitor (e.g., an anti -human PD-L1 antigen binding domain; (ii) a human protein transmembrane domain (e.g., a human CD2 transmembrane domain); and (iii) an intracellular domain comprising a human CD2 intracellular signaling domain.

[0329] In certain embodiments, the chimeric switch receptor comprisesSEQ ID NO: 1.

[0330] In some embodiments, the chimeric switch receptor comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 1.

[0331] In certain embodiments, the chimeric switch receptor comprises a leader sequence. In some embodiments, the leader sequence comprises SEQ ID NO: 2. In some embodiments, the leader sequence comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 2.

[0332] In certain embodiments, the chimeric switch receptor comprises a PD-1 LBD. In some embodiments, the PD-1 LBD comprises SEQ ID NO: 3. In some embodiments, the PD-1 LBD comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 3. Attorney Docket No. 046483 -7478WO 1(04048)

[0333] In certain embodiments, the chimeric switch receptor comprises a CD2 transmembrane domain. In some embodiments, the CD2 transmembrane domain comprises SEQ ID NO: 4. In some embodiments, the CD2 transmembrane domain comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 4.

[0334] In certain embodiments, the chimeric switch receptor comprises a CD2 intracellular signaling domain. In some embodiments, the CD2 intracellular signaling domain comprises SEQ ID NO: 5. In some embodiments, the CD2 intracellular signaling domain comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 5.

[0335] In certain embodiments, a nucleic acid of the present disclosure comprises a first nucleotide sequence and a second nucleotide sequence. The first and second nucleotide sequences may be separated a linker (i.e., a third nucleotide sequence). A linker for use in the present disclosure allows for multiple proteins to be encoded by the same nucleic acid sequence (e. ., a multi ci stronic or bicistronic sequence), which are translated as a polyprotein that is dissociated into separate protein components. In certain embodiments, the nucleic acid comprises from 5’ to 3’ the first nucleotide sequence, the linker, and the second nucleotide sequence. In certain embodiments, the nucleic acid comprises from 5’ to 3’ the second nucleotide sequence, the linker, and the first nucleotide sequence. In some embodiments, the linker comprises a nucleic acid sequence that encodes an internal ribosome entry site (IRES). As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art, including, without limitation, IRES obtainable from viral or cellular mRNA sources, e.g., immunoglobulin heavychain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast transcription factors Attorney Docket No. 046483 -7478WO 1(04048)

[0336] TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those of skill in the art would be able to select the appropriate IRES for use in the present invention.

[0337] In some embodiments, the linker comprises a nucleic acid sequence that encodes a selfcleaving peptide, also referred to as a ribosomal skipping sequence. As used herein, a “selfcleaving peptide” or “2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various selfcleaving or 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV), and porcine tescho virus- 1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively. Those of skill in the art would be able to select the appropriate selfcleaving peptide for use in the present invention.

[0338] In some embodiments, the construct includes a linker that optionally, further comprises a nucleic acid sequence that encodes a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-golgi and processes protein precursors before their secretion. Furin cleaves at the COOH- terminus of its consensus recognition sequence. Various furin consensus recognition sequences (or “furin cleavage sites”) are known to those of skill in the art. Those of skill in the art would be able to select the appropriate Furin cleavage site for use in the present invention.

[0339] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a Furin cleavage site and a 2A peptide. Examples include, without limitation, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and F2A, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and E2A, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and P2A, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and T2A. Those of skill in the art would be able to select the appropriate combination for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between the Furin cleavage site and the 2A peptide. In some embodiments, the linker comprises a Furin cleavage site 5’ to a 2A peptide. In Attorney Docket No. 046483 -7478WO 1(04048) some embodiments, the linker comprises a 2A peptide 5’ to a Furin cleavage site. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers (also known as GS linkers). Those of skill in the art would be able to select the appropriate spacer sequence for use in the present invention.

[0340] In some embodiments, a nucleic acid of the present disclosure may be operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art.

[0341] In certain embodiments, the nucleic acid encoding a CAR, TCR, or switch receptor is operably linked to a promoter. In certain embodiments, the promoter is a phosphoglycerate kinase- 1 (PGK) promoter.

[0342] For expression in a bacterial cell, suitable promoters include, but are not limited to, lad, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, e.g., promoter systems including Tet Activators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, Attorney Docket No. 046483 -7478WO 1(04048) soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

[0343] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used; see, e.g., Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by use of an Neri (p46) promoter; see, e.g., Eckelhart et al. Blood (2011) 117: 1565.

[0344] For expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter such as a GALI promoter, a GAL 10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac / tac hybrid promoter, a tac / trc hybrid promoter, a trp / lac promoter, a T7 / lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche- Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), a nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol. (1992) 10:888- 892); a sigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPL2 (see, e.g., WO96 / 17951); an actA promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70: 1087- 1096); an rpsM promoter (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); a tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. Attorney Docket No. 046483 -7478WO 1(04048)

[0345] (eds), Topics in Molecular and Structural Biology, Protein — Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984) 12:7035); and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and Plambda. Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25).

[0346] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences may also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0347] In some embodiments, the locus or construct or transgene containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et Attorney Docket No. 046483 -7478WO 1(04048) al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating irreversibly switched promoters and are well known in the art, see, e.g., Grindley el al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0348] In some embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR inducible expression cassette. In one embodiment, the CAR inducible expression cassette is used for the production of a transgenic polypeptide product that is released upon CAR signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine operably linked to a T-cell activation responsive promoter. In some embodiments, the cytokine operably linked to a T-cell activation responsive promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12.

[0349] A nucleic acid of the present disclosure may be present within an expression vector and / or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pST-Ki, pST-KiT, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0350] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors Attorney Docket No. 046483 -7478WO 1(04048) include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857- 2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319- 23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0351] Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

[0352] 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. Pat. No. 6,326,193).

[0353] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce the CAR, TCR, and / or switch receptor into an immune cell or precursor thereof (e.g., a T cell). Accordingly, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding for a CAR, TCR, and / or switch receptor. In some Attorney Docket No. 046483 -7478WO 1(04048) embodiments, the expression vector (e.g., lentiviral vector) will comprise additional elements that will aid in the functional expression of the CAR, TCR, and / or switch receptor encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding for a CAR, TCR, and / or switch receptor further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation-factor- 1 -alpha promoter (EF-la promoter). Use of an EF-la promoter may increase the efficiency in expression of downstream transgenes (e.g., a CAR encoding nucleic acid sequence). Physiologic promoters (e.g., an EF-la promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector (e.g., lentiviral vector) are known to those of skill in the art and may be incorporated into a vector of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises a non-requisite cis acting sequence that may improve titers and gene expression. One non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some embodiments a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector (e.g., lentiviral vector) of the present invention may Attorney Docket No. 046483 -7478WO 1(04048) comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, cPPT sequence, RRE sequence, 5’LTR, 3’ U3 deleted LTR’ in addition to a nucleic acid encoding for a CAR, TCR, and / or switch receptor.

[0354] Vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector may prevent viral transcription beyond the first round of viral replication. Consequently, a selfinactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors may greatly reduce the risk of creating a replication-competent virus.

[0355] In some embodiments, a nucleic acid of the present invention may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a CAR, TCR, and / or switch receptor of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a CAR, TCR, and / or switch receptor of the present disclosure.

[0356] In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.

[0357] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression Attorney Docket No. 046483 -7478WO 1(04048) is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include, without limitation, genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0358] In some embodiments, a nucleic acid of the present disclosure is provided for the production of a CAR, TCR, and / or switch receptor as described herein, e.g., in a mammalian cell. In some embodiments, a nucleic acid of the present disclosure provides for amplification of the CAR-, TCR-, and / or switch receptor-encoding nucleic acid.

[0359] Methods of Treatment

[0360] The modified immune cells (e.g., T cells) described herein may be included in a composition for immunotherapy for treating tumors or cancers, including solid tumors and nonsolid tumors. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified immune cells provided herein may be administered to a subject in need thereof.

[0361] In certain aspect, the invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified immune cells of the present invention. In one aspect, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the modified immune cells (i.e., a population of the modified immune cells) of the invention.

[0362] In some aspects, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of modified immune cells or precursor cells thereof comprising (i) a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and (ii) a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR). Attorney Docket No. 046483 -7478WO 1(04048)

[0363] Methods for administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive immune cell therapy methods are described, e.g., in US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PloS ONE 8(4): e61338; Lee et al., Int J. Mol Sci. (2021) 22(9):4590; Banerjee et al., JCO Clin Cancer Inform. (2021) 5:668-678; Robbins et al., Stem Cell Res Ther. (2021) 12(1):350; Wrona et al., Int J Mol Sci. (2021) 22(11 ):5899; Atrash and Moyo, Onco Targets Ther. (2021) 14:2185- 2201; Martinez Bedoya et al., Front Immunol. (2021) 12:640082; Morgan et al., Front Immunol. (2020) 11 : 1965; Chicaybam et al., Cancers (Basel) (2020) 12(9):2360; and Rafiq et al., Nat Rev Clin Oncol. (2020) 17(3): 147-167.

[0364] In some embodiments, the adoptive cell immunotherapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0365] In some embodiments, the adoptive cell immunotherapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0366] In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, Attorney Docket No. 046483 -7478WO 1(04048) the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0367] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0368] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0369] The immune cell of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell- mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the cells or pharmaceutical compositions of the invention include certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Exemplary cancers include but are not limited to B-cell malignancies such as B-cell lymphomas and leukemias and the like, as well as colorectal cancer, breast cancer, ovarian cancer, renal cancer, non-small cell lung cancer, melanoma, lymphoma, and hepatocellular cancers. The cancers may be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor. In certain embodiments, the cancer is a leukemia and / or a lymphoma. In certain embodiments, the cancer cells express CD 19.

[0370] In certain embodiments, the cancer is selected from the group consisting of a B-cell malignancy (e.g., a B-cell lymphoma or a leukemia), lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin’s lymphoma, non-Hodgkin’s Attorney Docket No. 046483 -7478WO 1(04048) lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophagus cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, and renal cancer.

[0371] The cells to be administered may be autologous, with respect to the subject undergoing therapy.

[0372] The administration of the 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, 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.

[0373] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

[0374] In some embodiments, the populations or sub-types of cells, such as CD8+and CD4+T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, Attorney Docket No. 046483 -7478WO 1(04048) administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4+to CD8+ratio), e.g., within a certain tolerated difference or error of such a ratio.

[0375] In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and / or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or subtype, or minimum number of cells of the population or sub-type per unit of body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and / or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD41to CD81cells, and / or is based on a desired fixed or minimum dose of CD4+and / or CD8+cells.

[0376] In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0377] In some embodiments, the dose of total cells and / or dose of individual sub-populations of cells is within a range of between at or about IxlO5cells / kg to about IxlO11cells / kg 104and at or about 1011cells / kilograms (kg) body weight, such as between 10?and 106cells / kg body weight, Attorney Docket No. 046483 -7478WO 1(04048) for example, at or about 1 x 105cells / kg, 1.5 x 103cells / kg, 2 x 103cells / kg, or 1 x 106cells / kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104and at or about 109T cells / kilograms (kg) body weight, such as between 105and 106T cells / kg body weight, for example, at or about 1 x 103T cells / kg, 1.5 x 105T cells / kg, 2 x 105T cells / kg, or 1 x 106T cells / kg body weight. In other exemplary embodiments, a suitable dosage range of cells for use in a method of the present disclosure includes, without limitation, from about IxlO5cells / kg to about IxlO6cells / kg, from about IxlO6cells / kg to about IxlO7cells / kg, from about IxlO7cells / kg about IxlO8cells / kg, from about IxlO8cells / kg about IxlO9cells / kg, from about IxlO9cells / kg about IxlO10cells / kg, from about IxlO10cells / kg about IxlO11cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO8cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO7cells / kg. In other embodiments, a suitable dosage is from about IxlO7total cells to about 5xl07total cells. In some embodiments, a suitable dosage is from about IxlO8total cells to about 5xl08total cells. In some embodiments, a suitable dosage is from about 1.4xl07total cells to about l .lxlO9total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl09total cells.

[0378] In some embodiments, the cells are administered at or within a certain range of error of between at or about 104and at or about 109CD4+and / or CD8+cells / kilograms (kg) body weight, such as between 103and 106CD4+and / or CD8+cells / kg body weight, for example, at or about 1 x 105CD4+and / or CD8+cells / kg, 1.5 x 105CD4+and / or CD8+cells / kg, 2 x 105CD4+and / or CD8+cells / kg, or 1 x 106CD4+and / or CD8 cells / kg body weight. In some embodiments, the cells are administered at or within a certain range of error of, greater than, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD4+cells, and / or at least about l x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD8+ cells, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106T cells. In some embodiments, the cells are administered at or within a certain range of error of between about 108and 1012or between about 1010and 1011T cells, between about 108and 1012or between about 1010and 1011CD4+cells, and / or between about 108and 1012or between about 1010and 1011CD8+cells. Attorney Docket No. 046483 -7478WO 1(04048)

[0379] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4+to CD8+cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1:5 and less than about 5: 1), or between at or about 1 :3 and at or about 3 : 1 (or greater than about 1:3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1 :5 (or greater than about 1 :5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1 : 1, 1 : 1, 1 : 1.1, 1 : 1.2, 1 : 1.3, 1 : 1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9: 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, or 1 :5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

[0380] In some embodiments, a dose of cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of cells is administered to a subject in need thereof by rapid intravenous infusion.

[0381] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject’s clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0382] In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy Attorney Docket No. 046483 -7478WO 1(04048) sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent.

[0383] In certain embodiments, the cells of the invention (e.g., the immune cell genetically modified to express a CAR) may be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PDl, anti-CTLA-4, or anti-PDLl antibody). For example, the cell may be administered in combination with an antibody or antibody fragment targeting, for example, PD-1 (programmed cell death protein 1). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or an antigenbinding fragment thereof. In certain embodiments, the cell may be administered in combination with an anti-PD-Ll antibody or antigen-binding fragment thereof. Examples of anti-PD-Ll antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi). In certain embodiments, the cell may be administered in combination with an anti-CTLA-4 antibody or antigen-binding fragment thereof. An example of an anti-CTLA-4 antibody includes, but is not limited to, Ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may also be used including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. Immune checkpoint modulators may be administered before, after, or concurrently with the cell comprising the CAR, TCR, and / or switch receptor. In certain embodiments, combination treatment comprising an immune checkpoint modulator may increase the therapeutic efficacy of a therapy comprising a cell of the present invention.

[0384] Following administration of the cells, the biological activity of the genetically modified cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an genetically modified or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the genetically modified cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, Attorney Docket No. 046483 -7478WO 1(04048) for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009); Herman et al. J. Immunological Methods, 285(1): 25-40 (2004); Kiesgen et al., Nat Protoc. (2021) 16(3): 1331- 1342; and Maldini et al., J Immunol Methods (2020) 484-485: 112830. In certain embodiments, the biological activity of the cells is measured by assaying expression and / or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[0385] In certain embodiments, the subject is provided a secondary treatment. Secondary treatments include but are not limited to chemotherapy, radiation, surgery, and medications.

[0386] In some embodiments, the subject can be administered a conditioning therapy prior to adoptive cell transfer immunotherapy (e.g., CAR T cell therapy). In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In preferred embodiments, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to CAR T cell therapy may increase the efficacy of the CAR T cell therapy. Methods of conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

[0387] In some embodiments, a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the immune cells of the invention. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0388] In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m2 / day. In some embodiments, the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m2 / day.

[0389] In some embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., Attorney Docket No. 046483 -7478WO 1(04048)

[0390] 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of about 30 mg / m2 / day.

[0391] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m2 / day over three days, and the dosing of fludarabine is 30 mg / m2 / day over three days.

[0392] Dosing of lymphodepletion chemotherapy may be scheduled on Days -6 to -4 (with a -1 day window, i.e., dosing on Days -7 to -5) relative to T cell (e.g., CAR-T, TCR-T, a modified T cell, etc.) infusion on Day 0.

[0393] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion 3 days prior to administration of the modified T cells. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion for 3 days prior to administration of the modified T cells.

[0394] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m2for 3 days.

[0395] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of 30 mg / m2for 3 days.

[0396] 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 compositions Attorney Docket No. 046483 -7478WO 1(04048) 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.

[0397] It is known in the art that one of the adverse effects following infusion of CAR T cells is the onset of immune activation, known as cytokine release syndrome (CRS). CRS is immune activation resulting in elevated inflammatory cytokines. CRS is a known on-target toxicity, development of which likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (constitutional symptoms and / or grade-2 organ toxicity) to severe CRS (sCRS; grade >3 organ toxicity, aggressive clinical intervention, and / or potentially life threatening). Clinical features include: high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation. Dramatic elevations of cytokines including interferon-gamma, granulocyte macrophage colony-stimulating factor, IL- 10, and IL-6 have been shown following CAR T-cell infusion. One CRS signature is elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations in clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with the CRS syndrome. The presence of CRS generally correlates with expansion and progressive immune activation of adoptively transferred cells. It has been demonstrated that the degree of CRS severity is dictated by disease burden at the time of infusion as patients with high tumor burden experience a more sCRS.

[0398] Accordingly, the invention provides for, following the diagnosis of CRS, appropriate CRS management strategies to mitigate the physiological symptoms of uncontrolled inflammation without dampening the antitumor efficacy of the genetically modified cells (e.g., CAR T cells). CRS management strategies are known in the art. For example, systemic corticosteroids may be administered to rapidly reverse symptoms of sCRS (e.g., grade 3 CRS) without compromising initial antitumor response.

[0399] In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the Food and Drug Administration-approved monoclonal antibody tocilizumab, also known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated near-immediate reversal of CRS. Attorney Docket No. 046483 -7478WO 1(04048)

[0400] CRS is generally managed based on the severity of the observed syndrome and interventions are tailored as such. CRS management decisions may be based upon clinical signs and symptoms and response to interventions, not solely on laboratory values alone.

[0401] Mild to moderate cases generally are treated with symptom management with fluid therapy, non-steroidal anti-inflammatory drug (NSAID) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability; with any hemodynamic instability, the administration of tocilizumab is recommended. The first-line management of CRS may be tocilizumab, in some embodiments, at the labeled dose of 8 mg / kg IV over 60 minutes (not to exceed 800 mg / dose); tocilizumab can be repeated Q8 hours. If suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with continued or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours or poor response to tocilizumab, may be treated with high- dose corticosteroid therapy, generally hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be administered high-dose corticosteroid therapy early in the course of the CRS. CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

[0402] Features consistent with Macrophage Activation Syndrome (MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007), coincident with clinical manifestations of the CRS. MAS appears to be a reaction to immune activation that occurs from the CRS, and should therefore be considered a manifestation of CRS. MAS is similar to HLH (also a reaction to immune stimulation). The clinical syndrome of MAS is characterized by high grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, and a decrease of circulating natural killer (NK) activity.

[0403] In one aspect, the invention includes a method of treating cancer in a subject in need thereof, comprising administering to the subject any one of the immune or precursor cells disclosed herein. Yet another aspect of the invention includes a method of treating cancer in a Attorney Docket No. 046483 -7478WO 1(04048) subject in need thereof, comprising administering to the subject an immune or precursor cell prepared by any one of the methods disclosed herein.

[0404] Sources of Immune Cells

[0405] In certain embodiments, a source of immune cells (e.g. T cells) is obtained from a subject for ex vivo manipulation and / or in vivo transduction. Sources of target cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow. For example, the source of immune cells may be from the subject to be treated with the immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject's bone marrow. Nondimiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. Methods for in vivo transduction of immune cells for CAR, TCR, and / or switch receptor expression are described, e.g., in Pfeiffer et al., EMBO Mol Med. (2018) 10(11): e9158; Weidner et al., Nat Protoc. (2021) 16(7):3210-3240; Frank et al., Blood Advances (2020) 4(22): 5702-5715; Nawaz et al., Blood Cancer J. (2021) 11(6): 119.

[0406] Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.

[0407] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), a macrophage, or a dendritic cell. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In an embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS Attorney Docket No. 046483 -7478WO 1(04048) cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g, a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0408] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa- associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any number of T cell lines available in the art, may be used.

[0409] In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and / or engineering them. In some embodiments, preparation of the genetically modified cells includes one or more culture and / or preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or genetically modified. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a Attorney Docket No. 046483 -7478WO 1(04048) biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0410] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0411] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0412] In some examples, cells from the circulating blood of a subject are obtained, e.g, by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood Attorney Docket No. 046483 -7478WO 1(04048) cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0413] In one embodiment, immune cells are obtained 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 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, 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.

[0414] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffmity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0415] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing Attorney Docket No. 046483 -7478WO 1(04048) the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0416] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0417] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker111811) of one or more particular markers, such as surface markers, or that are negative for (marker -) or express relatively low levels (markerlow) of one or more markers. For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and / or CD62L and / or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD 122, CD95, CD25, CD27, and / or IL7-Ra (CD 127). In some examples, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander). Attorney Docket No. 046483 -7478WO 1(04048)

[0418] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve longterm survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0419] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cell population and a CD8+ T cell sub-population, e.g., a subpopulation enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4- Attorney Docket No. 046483 -7478WO 1(04048) based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0420] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection.

[0421] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some Attorney Docket No. 046483 -7478WO 1(04048) embodiments, the stimulating agents include IL-2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0422] 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 an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.

[0423] 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 bound antibody.

[0424] 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 immune-adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface 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 CD14, CD20, CD1 lb, CD 16, HLA-DR, and CD8.

[0425] 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 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 concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used, n 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.

[0426] T cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the Attorney Docket No. 046483 -7478WO 1(04048) 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, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media.

[0427] The cells are then frozen to -80°C at a rate of 1°C 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.

[0428] 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.

[0429] In certain embodiments, T regulatory cells (Tregs) can be isolated from a sample. The sample can include, but is not limited to, umbilical cord blood or peripheral blood. In certain embodiments, the Tregs are isolated by flow-cytometry sorting. The sample can be enriched for Tregs prior to isolation by any means known in the art. The isolated Tregs can be cryopreserved, and / or expanded prior to use. Methods for isolating Tregs are described in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555,105, and U.S. Patent Application No. 13 / 639,927, contents of which are incorporated herein in their entirety.

[0430] Expansion of Immune Cells

[0431] Whether prior to or after modification of cells to express a CAR, the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Publication No. 20060121005. For example, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (c. ., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand Attorney Docket No. 046483 -7478WO 1(04048) that binds the accessory molecule is used. For example, T cells can be contacted with an anti- CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0432] Expanding T cells by the methods 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, 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.

[0433] Following culturing, the T cells can be incubated in cell medium in a culture 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 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 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.

[0434] 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.

[0435] Another procedure for ex vivo expansion cells is described in U.S. Pat. No. 5,199,942 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No. 5,199,942 can Attorney Docket No. 046483 -7478WO 1(04048) 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 with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.

[0436] 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0437] 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 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.

[0438] Each round of subculturing is referred to as a passage. When cells are subcultured, 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 example, a cultured cell population that has been passaged ten times may be referred to as a P10 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 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 between passaging.

[0439] 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 Attorney Docket No. 046483 -7478WO 1(04048) an appropriate media (e.g., Minimal Essential Media or RPM1 Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, 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, 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 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).

[0440] 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 that can stimulate CD28 is an antibody to CD28. A cell isolated by the methods disclosed herein can be expanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold, or more. In one embodiment, human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating T cells can be found in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555,105, contents of which are incorporated herein in their entirety.

[0441] 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 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 Attorney Docket No. 046483 -7478WO 1(04048) 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 cell function.

[0442] Pharmaceutical compositions and Formulations

[0443] Also provided are populations of immune cells of the invention, compositions containing such cells and / or enriched for such cells, such as in which cells expressing a CAR, TCR, and / or switch receptor make up at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[0444] Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0445] The term “pharmaceutical formulation” or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are Attorney Docket No. 046483 -7478WO 1(04048) typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0446] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0447] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, Attorney Docket No. 046483 -7478WO 1(04048) methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

[0448] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0449] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations. Attorney Docket No. 046483 -7478WO 1(04048)

[0450] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0451] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, c. ., by filtration through sterile filtration membranes.

[0452] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0453] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0454] EXPERIMENTAL EXAMPLES

[0455] 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 Attorney Docket No. 046483 -7478WO 1(04048) these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0456] Example 1 : A PD-1 :CD2 switch receptor provides CD2 signaling rescue in CAR T cells Materials and Methods

[0457] Cell lines and primary samples

[0458] Unless otherwise specified, all cell lines were cultured in R10 media (Roswell Park Memorial Institute medium 1640 (RPMI; Gibco; Cat#l 1875-085) supplemented with 10% fetal bovine serum (FBS, Gibco; Cat# 16140-071), 1% penicillin and 1% streptomycin (Gibco; Cat# 15140-163), 1% GlutaMAX supplement (Gibco; Cat# 35050-079, and 1% HEPES (Gibco; Cat# 15630-130)) in a 37°C incubator with 5% CO2. All cell lines were authenticated by short tandem repeat (STR) analysis and tested for mycoplasma using a MycoAlert Plus Mycoplasma Detection Kit (Lonza; Cat# LT07-710). Nalm6, Jurkat, OCLLy 18, and HEK293T cell lines were purchased from American Type Culture Collection (ATCC). TH20, a primary patient-derived xenograft model of T-cell acute lymphoblastic leukemia (T-ALL), was obtained as previously described (Diorio, et al., Blood. 2022;140(6):619-629). All cell lines were transduced with a lentivirus encoding click beetle green (CBG) and green fluorescent protein (GFP). Primary Sezary cells were provided by the clinical practices of Dr. Alain Rook. Primary acute myelogenous leukemia (AML) cells and other primary T-ALL cells were collected from patients treated at the Hospital of the University of Pennsylvania, as per IRB Protocol #855418.

[0459] CD 2 short guide RNA (sgRNA) optimization

[0460] CRISPR sgRNAs were designed using Benchling software (https: / / www.benchling.com). For both CD2 and CD58, sgRNA sequences were designed to target early exon sequences, and in vitro transcribed using the GeneArt Precision gRNA Synthesis Kit (Invitrogen; Cat# A29377) for screening. Cells were electroporated using the Lonza 4D-Nucleofector Core Unit. Primary T cells were electroporated using the P3 Primary Cell 4D-Nucleofector X Kit L (Lonza; Cat# V4XP-3024). For Cas9 and sgRNA delivery, the ribonucleoprotein (RNP) complex was initially formed by incubating 10 pg of TrueCut Cas9 Protein v2 (Lonza; Cat# A36499) with 10 pg of sgRNA for 10 minutes at room temperature. 5xl06cells were spun down at 300 g for 5 minutes and resuspended in 100 pL in the specified buffer. The RNP complex and 100 pL of Attorney Docket No. 046483 -7478WO 1(04048) resuspended cells were combined and electroporated using pulse code EO-115. After electroporation, the cells were incubated in standard media containing 20 ng / mL of supplemental cytokines IL-7 and IL-15 at a concentration of 2x 106cells / mL at 37°C. CD2 expression was subsequently monitored at each of the indicated days. After initial CD2 sgRNA screening, all subsequent experiments were performed using CD2gRNA8.

[0461] Lentivirus production

[0462] Replication-defective, third-generation lentiviral vectors were produced using HEK293T cells. Approximately 8xl06cells were plated in T150 culture vessels in standard culture media and incubated overnight at 37°C. 18-24 h later, cells were transfected using a combination of Lipofectamine 2000 (116 pL, Invitrogen; Cat#l 1668-019), pVSV / G or pCocal (7 pg), pRSV / Rev (18 pg), pGag / Pol (18 pg) packaging plasmids and 15 pg of expression plasmid. Lipofectamine and plasmid DNA were diluted in 4 mL Opti-MEM media (Gibco; Cat# 31985- 070) before transfer into lentiviral production flasks. At both 24 and 48 h following transfection, culture media was isolated and concentrated using high-speed ultracentrifugation (8,500 rpm overnight or 25,000 rpm for 2.5 hours).

[0463] CARs targeting CD2 were generated based on the antibody variable region sequences of monoclonal antibodies MEDI507, OKT11, Tl l-2, and TS2 18.1.1. Single-chain variable fragments (scFv) were designed in both orientations (from variable light chain to variable heavy chain and vice versa) with three glycine-serine-serine-serine linkers and synthesized at GenScript Biotech. After initial CD2 screening, all experiments were performed using MEDI507 in a light- to-heavy chain orientation. All CAR constructs were composed of an scFv, 4-1BB costimulatory domain, and CD3^ costimulatory domain, unless otherwise noted.

[0464] Manufacturing of primary human genome-engineered CAR T cells

[0465] Human T cells were procured through the University of Pennsylvania Human Immunology Core. CD4+and CD8+cells were combined at a 1 : 1 ratio and used for electroporation. CRISPR-Cas9 sgRNAs were generated using in vitro transcription, as described above. Mock KO cells were electroporated using the same procedure as described without the presence of an RNP complex. After electroporation, T cells were incubated at 37°C for 24 hours and subsequently activated using CD3 / CD28 Dynabeads (Gibco; Cat# 40203D) at a ratio of 3 Attorney Docket No. 046483 -7478WO 1(04048) beads / cell. The following day, CAR lentiviral vectors were added to stimulated cultures at a multiplicity of infection (MOI) of 1. Beads were removed at Day 5 of stimulation, and cells were counted every 2-3 days using a Moxi GO II (Orflo) until growth kinetics and cell size demonstrated they had rested from stimulation. All T cells were initially grown with 20 ng / mL of supplemental cytokines IL-7 and IL- 15 that was decreased to 0 ng / mL by the end of the expansion.

[0466] General mnltiparameter flow cytometry

[0467] Cells were resuspended in FACS staining buffer (phosphate buffered saline (PBS)+2% FBS) using one or more of the following antibodies: CD2 (UCHT2, 17H2L, or BLla), CD4 (OKT4), CD8 (SKI), CD45RA (2H4, ALB11), CCR7 (G043H7), PD1 (EH12.2H7), LAG3 (7H2C65), CD3 (UCHT1, OKT3), hCD45 (J33), mCD45 (30-F11), PDL1 (29E.2A3). CAR2 was detected by incubating cells an anti-G4S linker antibody conjugated to PE (CST; Cat# 71645S). CAR19 was detected using an anti-idiotype antibody provided by Novartis Pharmaceuticals. All changes in overall tumor or T cell counts reflected by absolute cell counts were determined using Flow-Count Fluorospheres (Beckman; Cat# 754053). Cell viability was established using ViaKrome 808 Fixable Viability Dye (Beckman; Cat# C36628) or LIVE / DEAD Fixable Violet Dead Cell stain (Invitrogen; Cat# L34955) and data were acquired on a CytoFlex LX Flow Cytometer (Beckman). All data analysis was performed using FlowJo 10.8.0 software (FlowJo, LLC).

[0468] Bioluminescence-based cell survival in vitro assays

[0469] Cell lines (Jurkat, Nalm6, and OCI-Lyl8) were engineered to express CBG, and cell survival was measured using bioluminescent quantification. D-luciferin potassium salt (Gold Biotechnology; Cat# 115144-35-9) was added to cell cultures (final concentration 15 pg / mL) and incubated at 37°C for 10 min. Bioluminescent signal was detected using a BioTek Synergy H4 imager, and signal was analyzed using BioTek Gen5 software. Percent cytotoxicity was calculated using a control of target cells without effectors. Attorney Docket No. 046483 -7478WO 1(04048)

[0470] Flow cytometry-based cell survival in vitro assays

[0471] Primary T-ALL, AML, or Sezary cells were stained with CellTrace Violet (CTV; Invitrogen; Cat# C34557) prior to plating with CAR T cells or control untransduced T cells. Reagents were used according to manufacturer protocol. After 48 h, cells were stained with ViaKrome 808 Fixable Viability Dye (Beckman; Cat# C36628) and analyzed by flow cytometry to determine absolute count of ViaKrome 808-negative and CTV+cells. Absolute cell counts were determined using Flow-Count Fluorospheres (Beckman; Cat# 754053). Percent cytotoxicity was calculated using a control of target cells without effectors.

[0472] Cell-to-cell avidity experiments

[0473] Z-Movi-compatible acoustofluidic chips were coated with poly-L-lysine for 3h prior to attaching a monolayer of tumor cells. CellTrace Far-Red (Invitrogen; Cat# C34564) labelled CAR T cells were allowed to incubate on the monolayer of tumor cells for 15 min, and then a ramping acoustic force was applied. Cell-detachment was analyzed using Imaged and R. Avidity experiments were conducted according to the manufacturers (LUMICKS™) instructions and recommendations.

[0474] Glass-supported planar lipid bilayer confocal imaging

[0475] Supported lipid bilayers were prepared by fusing small liposome droplets on clean glass coverslips as previously described (Guruprasad, et al., Nat Protoc. 2025 Jul 25, doi: 10.1038 / s41596-025-01208-x). Briefly, the liposome was trapped in a p-Slide VI 0.4 chamber (Ibidi, Germany, Cat# 50-305-784). Lipid bilayers were first blocked with 5% casein for 20 min and then incubated with 6.3 nM of streptavidin (Thermo Fisher) for 15 min. After washing with imaging buffer (HEPES-buffered saline), lipids were incubated with biotinylated CD 19 protein (R&D Systems, Cat# 9269-CD) conjugated with AlexaFluor 488, respectively, at room temperature for 30 min. After washing with the imaging buffer, lipid bilayers were blocked with 2.5 pM of D-biotin to saturate the streptavidin-binding sites. CAR T cells were then stimulated on lipid bilayers for 30 min at 37°C. Cells were then fixed in 4% paraformaldehyde for 10 min and then permeabilized in 0.5% Triton-XlOO and 10% normal donkey serum in PBS for 30 min at room temperature. Then, the cells were stained with primary antibodies against CD2 (ThermoFischer, Cat# 914-MSM3-P1ABX) and phosphorylated CD3 zeta (Abeam, Cat# Attorney Docket No. 046483 -7478WO 1(04048) ab68236) or CD19 (R&D Systems, Cat# AV19269). An Olympus FV3000 confocal microscope with 60x (NA 1.35) oil objective was used to obtain confocal image data. The fluorescence intensity is calculated as the sum of fluorescence at the synapse for individual cells.

[0476] In vivo immunodeficient mouse models

[0477] 6-10-week-old NOD-SCID-yc’ ‘ (MSG) mice were obtained from the Jackson Laboratory and maintained in pathogen-free conditions. All target cells were engineered to express CBG. Animals were injected via tail vein with IxlO6Jurkat, TH20, Nalm6, or Nalm6-PDL1- overexpressing cancer cells in 0.15 mL sterile PBS. For scRNAseq experiments, 5.0 x 106OCL Lyl8 were subcutaneously injected. After engraftment, IxlO6T cells (CAR+or an equal number of UTD control) were injected via tail vein in 0.15 mL sterile PBS, unless otherwise indicated. Animals were monitored for signs of disease progression and overt toxicity, such as xenogeneic graft-versus-host disease, as evidenced by >20% loss in body weight, loss of fur, diarrhea, conjunctivitis, and disease-related hind limb paralysis. Disease burdens were monitored over time using a Xenogen IVIS bioluminescent imaging system or the Lumina S5 imaging system for tumor flux.

[0478] Single cell RNA sequencing and analysis

[0479] Peripheral blood was collected from all mice and red blood cell lysis was performed. Samples were then processed as per manufacturer protocols using EasySep™ Mouse / Human Chimera Isolation Kit (Stem Cell Technologies; Cat# 19849) to negatively isolate human cells. After isolation, anti-hCD3, anti-hCD5, and anti-hCD2 antibodies were applied to stain for human T cells and subsequently positively sorted using BD FACSMelody™ Cell Sorter. Single-cell suspensions were loaded onto the 10X Genomics Chromium Controller. Gene expression libraries were prepared using the Chromium Single Cell 5' Reagent Kit with vl. l Chemistry (10X Genomics) according to the manufacturer's instructions. Libraries were sequenced using the Illumina NovaSeq 6000. Raw single-cell RNA sequencing data were pre-processed using the Cell Ranger software (version 5.0.1; 10X Genomics) transcript reads were mapped to the GRCh38 human reference genome. Filtered gene expression was processed using the Seurat package (version 4.0.1). To eliminate putative low-quality cells from the data set, the median absolute deviation (MAD)-based definition of outliers was used: cells with fewer than 200 Attorney Docket No. 046483 -7478WO 1(04048) expressed genes, or with an abnormally high number of UMI counts (> 3 MADs), or with high mitochondrial RNA expression (3 MADs) were discarded from downstream analysis.

[0480] Differential expression analyses and gene set enrichment analyses were computed using Seurat (Hao, et al., Cell. 2021;184(13):3573-3587.e29) and clusterProfiler (Wu, et al., Innovation (Cambridge (Mass.)). 2021;2(3): 100141), respectively, within R Studio. RNA Seq data were deposited in the Gene Expression Omnibus database.

[0481] CD2 patient validation cohorts

[0482] The flow cytometry archives (2002-2014) at the Children’s Hospital of Philadelphia (CHOP) were reviewed to identify cases of T-ALL (approved by CHOP) in the pediatric and young adult age group (0-25 years). Diagnosis of T-ALL was confirmed per current WHO criteria (Arber, et al., Blood. 2016;127(20):2391-405). Archived flow cytometric histograms that were saved as PDFs were reviewed. Flow cytometric data was collected using Beckman Coulter FC 500 Series flow cytometers in a College of American Pathologists (CAP) accredited clinical flow cytometry laboratory. Bead controls, titration of newer antibody lots and daily comparison of instruments were performed. One million cells / microliter single cell suspensions of bone marrow, peripheral blood or tissue were incubated with 5 color antibody panels including CD45- PE-Cy7, CD4-PE, CD8-FITC, CD7-PE-Cy5.5, CD3-ECD, CD2-FITC, CD56-PE, B cell, myeloid markers, and isotype control antibodies (Beckman Coulter, CA). Twenty thousand events were collected. Data analysis was performed using FCS Express (De Novo software, CA). Negative gates were based on isotype controls. Immature blasts were identified using CD45 vs. SSC gating. T cell markers CD2, sCD3, CD5 and CD7 were assessed on CD45dim blasts. Normal CD45bright mature T cells served as controls for assessment of antigen.

[0483] The phenotypic profile of peripheral T cell lymphomas from 91 patients diagnosed and treated at the European Institute of Oncology was analyzed via immunohistochemistry for CD2, CD3, CD5, CD7 expression by a board-certified hematopathologist. T-ALL CITE-seq data from patients enrolled to AALL0434 was obtained from Tan, et al. Res. Sq. 2023, doi: 10.21203 / rs.3.rs-3487715 / vl). CITE-seq data was normalized using CLR normalization across cells (margin = 2). Data was integrated using the IntegrateLayers function in Seurat v5 using RPC A and visualized on a UMAP using the top 30 principal components (Hao, et al. Nat Biotechnol. 2024;42(2):293-304). Attorney Docket No. 046483 -7478WO 1(04048)

[0484] CD58 immunohistochemistry of primary B cell lymphomas

[0485] CD58 immunohistochemical stain was performed on selected patient samples. Five- micron sections of formalin-fixed paraffin-embedded tissue were stained using antibody against CD58 at 1 : 100 dilution (R&D systems, Cat# AF1689). Staining was done on a Leica Bond- IIITM instrument using the Bond Polymer Refine Detection System (Leica Microsystems DS9800) and Goat IgG VisUCyte HRP Polymer (R&D Systems, Cat# VC004-25). Heat-induced epitope retrieval was done for 20 minutes with ER2 solution (Leica Microsystems AR9640). All the experiments were done at room temperature. Slides were washed three times between each step with bond wash buffer or water. Positive staining was visualized and scoring of percentage of tumor cells and intensity of staining were determined by at least two hematopathologists. Hematopathologists were blinded to patient outcomes and categorization at the time of immunohistochemical evaluation. The percentage of tumor stains staining was determined by evaluating previous H&E and immunohistochemical stains performed at initial diagnosis. Positive staining was defined as clear membranous staining present. Intensity was determined by evaluating immunohistochemical stain controls with the strongest intensity membranous staining interpreted as 3+ and the weakest intensity staining observed interpreted as 1+ staining. Variable or non-specific staining was scored as negative. The final H-score was calculated by multiplying the intensity of staining by the percentage of tumor cells staining.

[0486] Statistical analysis

[0487] Data were visualized and analyzed using Prism 10 software (GraphPad). All results are represented as either individual values or as mean values ± standard error of the mean (SEM) unless otherwise noted. All comparisons between two groups were performed using two-tailed unpaired Student’s t-test. Comparisons between more than two groups were performed by oneway analysis of variance (ANOVA) with Tukey correction for multiple comparisons. In analyses where multiple groups were compared at multiple time points / ratios, two-way ANOVA was performed. Survival data were analyzed using the Log-Rank (Mantel-Cox) test unless otherwise noted. The p values were denoted with asterisks as follows: * p < 0.05, ** p < 0.01, *** p < 0.00!, **** p < 0.0001. Attorney Docket No. 046483 -7478WO 1(04048)

[0488] Results

[0489] CD2 is a transmembrane glycoprotein of the immunoglobulin supergene family that is uniformly expressed on mature T-, NK-, and T-cell progenitors (Binder, et al., Front Immunol. 2020; 1 l(June): 1-14). Notably, CD2 has high and uniform expression across the spectrum of T cell malignancies and represents a highly attractive target for cellular immunotherapies. Human CD2 preferentially engages with CD58 (LFA-3) present on antigen-presenting and target cells to facilitate immune synapse formation (Springer, et al., Nature. 1990;346(6283):425-434; Demetriou, et al., Nat Immunol . 2020;21(10):1232-1243; Wild, et al., J Exp Med.

[0490] 1999; 190(l):31-42). CD2:CD58 binding primarily occurs within the peripheral supram olecul ar activation cluster (pSMAC) in concert with other molecules, such as PD-1 :PD-L1 to support T cell receptor (TCR) complex engagement with peptide-major histocompatibility complex (pMHC) (Zhang, et al., Front Immunol . 2021;12; Capitani, et al., Front Immunol . 2022;13;

[0491] Siokis, et al., iScience. 2021 ;24(10): 103100). Intracellular CD2 serves as a costimulatory molecule that contributes to T cell activation, proliferation, and effector function in an MHC- dependent manner (Leitner, et al., The Journal of Immunology . 2015;195(2):477-487; Pettmann, et al., Elife. 2021;10; Kaizuka, et al., Journal of Cell Biology. 2009; 185(3):521—534). Loss of CD2:CD58 engagement in endogenous T cells is associated with cancer immune evasion and poor anti-tumor responses (Ho, et al., Cancer Cell. 2023 ;41(7): 1207-1221. el2). However, as CAR signaling is mediated in an MHC-independent manner, an analogous role of CD2 in CAR T cell immunobiology has not been clearly established to date.

[0492] Previously, it was shown that anti-CD2 CAR T cells with endogenous CD2 knock-out (CART2) were highly efficacious against CD2-expressing neoplasms and eliminated CAR T cell fratricide (WO 2020 / 132327 Al). Using this optimized product, high in vitro and in vivo efficacy against patient-derived models of T cell leukemia was observed. However, when anti-CD19 CAR T cells (CART 19) against B-cell lymphoma were used, it was observed that CD2 deletion in T cells or CD58 deletion in tumor cells attenuated CAR T cell activity. As demonstrated herein, the CD2:CD58 deficiency is shown to be reversible with intracellular CD2 signaling rescue provided by a PD-1 :CD2 switch receptor engineered into anti-CD19 CAR T cells. Attorney Docket No. 046483 -7478WO 1(04048)

[0493] CD2 deletion decreases the anti-tumor function of CAR T cells

[0494] Potent anti-tumor efficacy of CD2KO CART2 in T cell leukemia and lymphoma models was shown. Thus, the impact of CD2 deletion in the broader CAR T cell space, and the relative contribution of CD2 to CAR T cell efficacy, was investigated herein. Given that CD2+CART2 cannot be manufactured due to fratricide, anti-CD19 CAR T cells were used to test CD2 immunobiology. First, it was observed that anti-CD19 CAR T cells showed no significant differences in T cell population doubling throughout manufacturing in the absence of CD2 (FIG. 2A). Next, the cytotoxicity of CD2KO CART 19 (with a 4- IBB costimulatory domain) was compared to that of CD2WT CART19 using a B-ALL model (Nalm6). While CD2KO CART19 retained some cytotoxic effector activity as compared to CD2WT CART 19 at higher E:T ratios, their effect was reduced as compared to CD2WT CART19 at a low E:T ratio over 72 hours of co-culture (FIG. 1A). A more pronounced effect was seen in vivo as expected (FIG. IB). Indeed, CD2KO CART 19 treated mice failed to adequately control Nalm6 progression as compared to CD2WT CART19-treated mice (FIG. 1C, FIG. 2B). Poor tumor control in the CD2KO CART 19 treated mice was confirmed with an independent replicate CART 19 donor and correlated with a worse median overall survival (mOS: Mock UTD: 14 days, CD2KO UTD: 14 days, CD2KO CART19: 28 days, CD2WT CART19: 38 days; p < 0.05 between CD2KO CART19 vs. CD2WT CART19, Mantel-Cox) (FIG. ID, FIG. 2B). Death was attributable, in all cases, to leukemic relapse and not graft-versus-host disease (FIG. 2C).

[0495] Next, plasma cytokine secretion in CD2-deleted CART 19 was investigated. Using a multiplexed human cytokine quantification panel, a generalized reduction in CAR T-mediated cytokine release was found in plasma isolated from CD2KO CART19 mice as compared to CD2WT CART19 treated mice, with a specific reduction in T cell activating cytokines, such as IFN-y, IL-2, sILR2oc, IL-5, and CXCL10 / IP (FIG. 2D). Since CD2 signaling is known to synergize with CD28 to support TCR-dependent cytotoxic activity in endogenous T cells (Leitner, et al., The Journal of Immunology . 2015;195(2):477-487; Pettmann, et al., Elife. 2021; 10), whether the presence of CD28 as a CAR costimulatory domain would overcome the lack of CD2 signaling and support long-term efficacy of CD2-deficient CAR T cells was also tested. To this goal, NSG mice engrafted with Nalm6 were injected with CD28-costimulated CART19 cells, with and without CD2 knockout. Again, the results show that CD2KO CD28- Attorney Docket No. 046483 -7478WO 1(04048) costimulated CART19 underperformed CD2WT CART19 in both leukemia control and overall survival (FIG. 2E).

[0496] Lack ofCD2 on CAR T cells leads to reduced T cell activation and decreased effector function

[0497] Having demonstrated that CD2KO decreases CAR T cell function, the mechanism behind this effect was explored. First, the role CD2 plays in organizing a CAR T cell immune synapse (IS) was examined. Using confocal immunofluorescent microscopy, the colocalization of CD2 to the CD19:CART19 immune synapse in a CD 19-lipid bilayer model was visualized. CD2 localized to the CD19:CART19 immune synapse in activated CD2WT CART19 cells, however, its absence did not prevent CD19:CART19 engagement (FIG. IE). It was also observed that F- actin accumulation was highly significantly reduced in CD2KO CART19 immune synapse. F- actin is an important cytoskeletal protein known to facilitate intracellular T cell activation in conjunction with the TCR (Binder, et al., Front Immunol. 2020; 1 l(June): 1-14; Badour, et al., Immunity. 2003; 18(1): 141— 154; Dustin, et al., Nat Immunol . 2000;l(l):23-9; Hutchings, et al., J Biol Chem. 2003;278(25):22396-403). Given that defects in immune synapse formation were shown, its effect on the CAR T celktumor cell interaction were next studied directly. To this end, cellular avidity was quantified using the Z-Movi platform. The results show that loss of CD2 correlated with decreased, but not complete loss, of CAR T cell Tumor target avidity in both endogenous T cells (Mock UTD vs. CD2KO UTD) and CART 19 (Mock CART 19 vs. CD2KO CART 19) (FIG. IF). Taken together, these data strongly indicate that human CD2 mediates, in part, assembly of a CAR T immune synapse in an MHC -independent manner, which in turn affects optimal CAR T cell binding to tumor cells.

[0498] To characterize the effects of this defective immune synapse and reduced avidity in CD2KO vs. CDWT CART 19 cells, single-cell RNA sequencing was conducted on human CAR T cells harvested from an in vivo model. Mice were first engrafted subcutaneously with 5 x 106CD 19-positive B cell lymphoma OCI-Lyl8 on day -10 and subsequently treated at day 0 with 5 x 106CD2WT or CD2KO CART 19 T cells on day 0 by intravenous injection (FIG. 3A, FIG. 4A). The study was terminated on day 16 when the CART 19 groups were at peak anti -tumor activity. T cells were harvested from peripheral blood, positively selected for human T cell antigens (CD3, CD2, and CD5) and subjected to single-cell RNA sequencing. Several clusters Attorney Docket No. 046483 -7478WO 1(04048) were identified based on gene expression; in particular, clusters 6 and 7 were particularly noteworthy as they were exclusive to CD2KO CART19 cells and comprised predominantly CD4+and CD8+effector memory T cells (FIG. 3B, FIG. 4B, FIG. 4C). Specific to cluster 7 were CD8+T cells (G7MB, GZMH, PRF1 overexpression) enriched in genes associated with T cell exhaustion (FIG. 3C), such as LAG3 (Andrews, et al., Immunol Rev. 2017;276(l):80-96; Grosso, et al., J Immunol. 2009; 182(11):6659-69; Workman, et al. J Immunol.

[0499] 2002;169(10):5392-5) and SLAMF7 (Awwad, et al., Leukemia. 2021;35(9):2602-2615; O’Connell, et al., J Immunol. 2021;206(l): 193-205). Gene set enrichment analysis of gene ontology on clusters 6 (NES: -2.00) and 7 (NES: -1.517) indicated that T cell activation was depleted in CD2 KO CART19 cells (FIG. 3D). Moreover, a profound reduction of key genes representative of tumor-reactive cytotoxic T cells was observed that were conserved in both clusters 6 and 7 (FIG. 3E), including TCF7 (Germar, et al., Proc Natl Acad Sci USA.

[0500] 2011;108(50):20060-5; Jeannet, et al., Proc Natl A cad Sci USA. 2010;107(21):9777-82), LEF1 (Travis, et al., Genes Dev. 1991;5(5):880-94; Okamura, et a\ . Immunity. 1998;8(1): 11-20), CCR7 (Forster, et al., Cell. 1999;99(l):23-33; Ueno, et al. Immunity. 2002;16(2):205-18), and IGOS (Dong, et al., Nature. 2001;409(6816):97-101). These findings strongly indicate that CD2- mediated signaling is also necessary in CAR T cells to support a pro-inflammatory, tumor reactive T cell phenotypes.

[0501] CD58 plays a critical role in CD2-mediated CAR T cell stimulation and anti-tumor effects

[0502] Because CD2 signaling was shown to contribute to CAR T cell efficacy, the role of CD58 was next studied. CD58 is the predominant ligand of human CD2, expressed on hematopoietic and non-hematopoietic cells in certain tumors (Krensky, et al. J Immunol. 1983; 131(2):611— 616). First, whether CD58 deletion was significantly associated with CAR T cell resistance was investigated by analyzing a short-term, unbiased, genome-wide CRISPR-Cas9 knock-out screen of Nalm6 combined with CART19 cells. This platform serves as an exploratory, loss-of-function model of tumor-specific genes that regulate primary CART19 resistance (Singh, et al., Cancer Discov. 2020;10(4):552-567). The results show a highly significant enrichment of a human CD58-specific gRNA (leading to the knockout of CD58) correlating with CART19 resistance (FIG. 5A). Furthermore, to validate this on a clinical level, bulk RNA sequencing was performed Attorney Docket No. 046483 -7478WO 1(04048) on diagnostic biopsies of 17 patients with diffuse large B cell lymphoma (DLBCL) prior to investigational CART19 treatment at the University of Pennsylvania (NCT02030834). The results show a statistically significant overall survival advantage in patients stratified to the highest CD58 tertile (mOS: 26.2 months, n=12) as compared to patients in the lowest tertiles (7.9 months, n=5, p=0.0083, Wilcox rank sum) (FIG. 5B). Complementary, no significant differences in CD2 expression on CAR T cells was observed between responsive and refractory patients (n=28) infused with tisagenlecleucel (data not shown). Thus, tumor-associated CD58 aberrancies were hypothesized to influence CART 19 responses.

[0503] To gain clinical insight of the role of CD58 in CART19 immunotherapy, an assessment was performed of CD58 staining in biopsies of relapsed and / or refractory B cell lymphomas taken before (n = 27) and after (n = 25) commercial CART 19 administration at the University of Pennsylvania. All pre-CART19 biopsies were obtained to confirm the diagnosis of an advanced B cell lymphoma and all post-CART19 biopsies were obtained at clinical or radiographic disease progression. The results show a generalized reduction in overall CD58 h-score by immunohistochemistry (IHC) post-CART19 in relapsed patients (H-score: 157.0 ± 18.3) as compared to pretreatment (H-score: 184.7 ± 16.4). Pre-CART19 CD58 h-score was higher in patients who achieved a durable complete response exceeding 1 year after CART 19 treatment as compared to those with a complete response less than 1 year or no response after CART 19 treatment (Long CR: 224.5 ± 21.1, Short CR: 183.0 ± 20.0, Refractory / Relapsed: 176.1 ± 28.0). CD58 IHC was further predictive in that pre-CART19 biopsies in the lowest H-score tertile yielded worse durations of CART 19 responses (FIG. 5C). Furthermore, in patients where paired biopsies were available, CD58 IHC expression was either maintained or reduced at relapse in 78.6% (11 / 14) of cases (FIG. 5D). This trend was independent on B cell lymphoma subtype or commercial CAR T cell product infused (FIG. 6A).

[0504] Next, experiments were performed to test whether CD58 deletion on tumor cells would similarly attenuate CART 19 activity, as was observed with CD2 deletion on CART 19 cells. Specifically, CD58 knock-out (CD58KO) Nalm6 and OCI-Lyl8 (FIG. 6B) cell lines were generated and short-term cytotoxicity assays were performed. The results show that CD58KO on Nalm6 and OCI-Lyl8 each attenuated cytotoxicity as compared to CD58 wild-type (CD58WT) controls against CART19 (FIG. 5E). Mechanistically, similar to what was observed for CD2, Attorney Docket No. 046483 -7478WO 1(04048)

[0505] CD58 deletion was associated with poor CART19 avidity (FIG. 5F). Collectively, these data establish the need for CD2:CD58 engagement for optimal CART19 efficacy.

[0506] A PD-1 :CD2 switch receptor rescues the lack of CD 2 engagement on CART 19

[0507] The results shown herein highlight the need to develop translational strategies to overcome the lack of CD2 signaling (either CD2 deletion on CAR T cells or CD58 aberrations on tumor cells) to ensure optimal synapse formation, costimulation, and ultimately anti-tumor effect of CAR T immunotherapy. A previous strategy involved incorporating the intracellular CD2 signaling domain (iCD2) in cis with a costimulatory domain as a third-generation CART 19 (CART19-iCD2) (FIG. 4C). Although this approach was successful in enhancing in vitro cytotoxicity at low effector-to-target cell ratios (data not shown), CART19-iCD2 cells were unable to recapitulate these results in an in vivo model against CD58KO Nalm6 (data not shown), possibly related to the membrane-distal arrangement of CD2 within the CAR construct. Interestingly, this is in accord to what has been shown using an anti-CD22 CAR T cell model system (Majzner, et al., Blood. 2020;136(Supplement 1):53- 54).

[0508] As such, it was contemplated herein that a novel in trans switch receptor comprising a membrane-proximal intracellular CD2 domain would be able to provide CD2 signaling and thereby activate CAR T cells that lack CD2:CD58 engagement (FIG. 5G). PD-1 was selected as the extracellular domain of this switch receptor due to its close proximity to CD2 in T cell synapses (Demetriou, et al., Nat Immunol. 2020;21(10): 1232-1243). Thus, it was hypothesized that a PD-1 :CD2 switch receptor would simultaneously engage PD-L1 on tumor cells to trigger CD2 intracellular signaling along with CD19:CART19 engagement. Using a P2A lentiviral plasmid, high co-expression of the PD-1 :CD2 switch receptor with the anti-CD19 CAR was obtained in CART19 in tandem with CD2 deletion (FIG. 5H, FIG. 6C). The addition of the PD- 1:CD2 switch receptor did not affect CART19 manufacturing kinetics (FIG. 6C). Next, PDL-1- expressing Nalm6 were engrafted in NSG mice. Leukemic growth following treatment with CD2KO CART19 or CD2KO PD-1 :CD2 CART19 cells was compared. The results show improved tumor control in CD2KO PD-1 :CD2 CART 19 treated mice, which correlated with a significant improvement in overall survival (FIG. 51). These data strongly indicate that perturbations in the CD2:CD58 signaling axis can be circumvented by CD2 signaling rescue as exemplified using co-expression of a PD-1 :CD2 switch receptor in CART19. Attorney Docket No. 046483 -7478WO 1(04048)

[0509] Example 2: Harnessing the CD2 axis to broaden and enhance the efficacy of CAR T cell therapies

[0510] Materials and Methods

[0511] Overview

[0512] Nalm6, Jurkat, OCI-Lyl8, HEK293T (ATCC), and the TH20 T-ALL PDX (39) were maintained in RPMI-based R10; all lines were STR-authenticated and mycoplasma-free. Primary Sezary cells were from Dr. Alain Rook, and AML / T-ALL samples were collected at Penn under IRB approval (#855418). Genome-edited CAR T cells were generated from CD47CD8+T cells (1 : 1) from the Penn Human Immunology Core (IRB #705906), electroporated with Cas9-sgRNA RNPs targeting CD2 or CD58 (Table 1 and Table 2), cultured in IL-7 / IL-15, activated with CD3 / CD28 Dynabeads, and transduced with lentiviral CAR vectors (MOI 1). Murine T cells were engineered similarly, and lentiviruses were produced in HEK293T cells, concentrated, and designed with 4-lBB / CD3(^ signaling; the MEDI507 orientation was used for definitive studies (Table 3). CAR expression was detected by flow cytometry using anti-idiotype antibodies (CAR19) or anti-G4S linker antibodies (CAR2, murine CAR19); a list of specific antibodies is provided in the Supplemental Materials. Cytotoxicity was quantified by luciferase-based assays or CellTrace Violet dilution, while proliferation and metabolism were assessed by flow cytometry and Seahorse XF96 profiling. Avidity was measured with Z-Movi acoustofluidic chips (LUMICKS™). Jurkat triple-reporter assays (E:T 1: 1) evaluated NF AT, NF-KB, and AP-1 activity after co-culture with CAR constructs ± full-length or truncated switch receptors. Imaging assays used supported lipid bilayers functionalized with biotinylated CD 19 to stimulate CAR T cells, with synapse formation visualized on an Olympus FV3000 confocal microscope(40)or analyzed in NALM6-GFP co-cultures by Leica Stellaris. In vivo experiments were performed in NSG or BALB / c mice bearing human or murine tumors, with CAR T infusion after engraftment or cyclophosphamide conditioning, and disease monitored by IVIS or caliper measurements (Guruprasad, et al., Nat Protoc. 2025 Jul 25, doi: 10.1038 / s41596-025-01208-x). Single-cell RNA sequencing was performed using 10X v2 chemistry, sequenced on NovaSeq, and analyzed in Seurat v5 with clusterProfiler (Hao, et al., Cell. 2021 Jun 24;184(13):3573-3587.e29; Szabo, et al., Nat Common. 2019 Oct 17;10(l):4706; Wu, et al., clusterProfder 4.0: A universal enrichment tool for interpreting omics data. Innovation (Cambridge (Mass)). 2021 Aug 28;2(3): 100141). Clinical cohorts included pediatric / young adult T-ALL (CHOP, 2002-2014), Attorney Docket No. 046483 -7478WO 1(04048)

[0513] PTCL from the European Institute of Oncology, and CITE-seq biobanking studies (Arber, et al., Blood. 2016 May 19;127(20):2391-405; Tan, et al., Res Sq. 2023 Oct 30; Dunsmore, et al., J Clin Oncol. 2020 Oct l;38(28):3282-93; Hao, et al., Nat Biotechnol. 2024 Feb;42(2):293-304). CD58 immunohistochemistry was scored by blinded hematopathologists.

[0514] Table 1: CD2 and CD58 gRNA sequences tested

[0515] Table 2: Screening of gRNAs for Murine CD2 Knockout Table 3: CAR2 scFv antibody clones Attorney Docket No. 046483 -7478WO 1(04048) Attorney Docket No. 046483 -7478WO 1(04048)

[0516] Cell lines and primary samples

[0517] Unless otherwise specified, all cell lines were cultured in RIO media (Roswell Park Memorial Institute medium 1640 (RPMI; Gibco; Cat#l 1875-085) supplemented with 10% fetal bovine serum (FBS, Gibco; Cat# 16140-071), 1% penicillin and 1% streptomycin (Gibco; Cat# 15140-163), 1% GlutaMAX supplement (Gibco; Cat# 35050-079, and 1% HEPES (Gibco; Cat# 15630-130)) in a 37°C incubator with 5% CO2. All cell lines were authenticated by short tandem repeat (STR) analysis and tested for mycoplasma using a MycoAlert Plus Mycoplasma Detection Kit (Lonza; Cat# LT07-710). Nalm6, Jurkat, OCI-Ly 18, and HEK293T cell lines were purchased from American Type Culture Collection (ATCC). TH20, a primary patient-derived xenograft model of T-cell acute lymphoblastic leukemia (T-ALL), was obtained as previously described (Diorio, et al., Blood. 2022 Aug 11 ; 140(6):619— 29). All cell lines were transduced with a lentivirus encoding click beetle green (CBG) and green fluorescent protein (GFP). Primary Sezary cells were provided by the clinical practices of Dr. Alain Rook. Primary acute myelogenous leukemia (AML) cells and other primary T-ALL cells were collected from patients treated at the Hospital of the University of Pennsylvania, as per IRB Protocol #855418.

[0518] CD2 short guide RNA (sgRNA) optimization

[0519] CRISPR sgRNAs were designed using Benchling. For CD2, CD58, and the murine CD2, sgRNA sequences were designed to target early exon sequences, and in vitro transcribed using the GeneArt Precision gRNA Synthesis Kit (Invitrogen; Cat# A29377) for screening. Cells were electroporated using the Lonza 4D-Nucleofector Core Unit. Primary T-cells (human or mouse) were electroporated using the P3 Primary Cell 4D-Nucleofector X Kit L (Lonza; Cat# V4XP- 3024). For Cas9 and sgRNA delivery, the ribonucleoprotein (RNP) complex was initially formed by incubating 10 pg of TrueCut Cas9 Protein v2 (Lonza; Cat# A36499) with 10 pg of sgRNA for 10 minutes at room temperature. 5xl06cells were spun down at 300 x g for 5 minutes and resuspended in 100 pL in the specified buffer. The RNP complex and 100 pL of resuspended cells were combined and electroporated using pulse code EO-115. After electroporation, human T-cells were incubated in standard media containing 20 ng / mL of supplemental cytokines IL-7 and IL-15 at a concentration of 2* 106cells / mL at 37°C. For mouse T-cells were used mouse-IL7 and human-IL2 at a concentration of 2x 106cells / mL at 37°C. CD2 or murine CD2 expression was subsequently monitored at each of the indicated days. After initial CD2 sgRNA screening, Attorney Docket No. 046483 -7478WO 1(04048) all subsequent experiments were performed using CD2gRNA8 for the human CD2 and CD2gRNA5 for mouse CD2.

[0520] Lentivirus production

[0521] Replication-defective, third-generation lentiviral vectors were produced using HEK293T cells. Approximately 8xl06cells were plated in T150 culture vessels in standard culture media and incubated overnight at 37°C. 18-24 h later, cells were transfected using a combination of Lipofectamine 2000 (116 pL, Invitrogen; Cat# 11668-019), pVSV / G or pCocal (7 pg), pRSV / Rev (18 pg), pGag / Pol (18 pg) packaging plasmids and 15 pg of expression plasmid. Lipofectamine and plasmid DNA were diluted in 4 mL Opti-MEM media (Gibco; Cat# 31985- 070) before transfer into lentiviral production flasks. At both 24 and 48 h following transfection, culture media was isolated and concentrated using high-speed ultracentrifugation (8,500 rpm overnight or 25,000 rpm for 2.5 hours).

[0522] CARs targeting CD2 were generated based on the antibody variable region sequences of monoclonal antibodies MEDI507, OKT11, T11-2, and TS2 18.1.1. Single-chain variable fragments (scFv) were designed in both orientations (from variable light chain to variable heavy chain and vice versa) with three glycine-serine-serine-serine linkers and synthesized at GenScript Biotech. After initial CD2 screening, all experiments were performed using MEDI507 in a light- to-heavy chain orientation. All CAR constructs were composed of an scFv, 4- IBB costimulatory domain, and CD3z costimulatory domain, unless otherwise noted.

[0523] Manufacturing of primary human genome-engineered CAR T-cells

[0524] Human T-cells were procured through the University of Pennsylvania Human Immunology Core (IRB #705906). CD4+and CD8+cells were combined at a 1 : 1 ratio and used for electroporation. CRISPR-Cas9 sgRNAs were generated using in vitro transcription, as described above. Mock KO cells were electroporated using the same procedure as described without the presence of an RNP complex. After electroporation, T-cells were incubated at 37°C for 24 hours and subsequently activated using CD3 / CD28 Dynabeads (Gibco; Cat# 40203D) at a ratio of 3 beads / cell. The following day, CAR lentiviral vectors were added to stimulated cultures at a multiplicity of infection (MOI) of 1. Beads were removed at Day 5 of stimulation, and cells were counted every 2-3 days using a MoxiGO II (Orflo) until growth kinetics and cell size demonstrated they had rested from stimulation. All T-cells were initially grown with 20 Attorney Docket No. 046483 -7478WO 1(04048) ng / mL of supplemental cytokines IL-7 and IL-15 that were decreased to 0 ng / mL by the end of the expansion.

[0525] General multiparameter flow cytometry

[0526] Cells were resuspended in FACS staining buffer (phosphate buffered saline (PBS)+2% FBS) using one or more of the following antibodies: CD2 (UCHT2, 17H2L, or BLla), CD4 (OKT4), CD8 (SKI), CD45RA (2H4, ALB11), CCR7 (G043H7), PD1 (EH12.2H7), LAG3 (7H2C65), CD3 (UCHT1, OKT3), hCD45 (J33), mCD45 (30-F11), PD-L1 (29E.2A3), mouse CD2 (RM2-5), mouse PD-1 (EH12.2H7), CAR2 and murine CAR19 were detected by incubating cells with an anti-G4S linker antibody conjugated to PE (CST; Cat# 71645S). Human CAR19 was detected using an anti -idiotype antibody provided by Novartis Pharmaceuticals. All changes in overall tumor or T-cell counts reflected by absolute cell counts were determined using Flow-Count Fluorospheres (Beckman; Cat# 754053). Cell viability was established using ViaKrome 808 Fixable Viability Dye (Beckman; Cat# C36628) or LIVE / DEAD Fixable Violet Dead Cell stain (Invitrogen; Cat# L34955), and data were acquired on a CytoFlex LX Flow Cytometer (Beckman). All data analysis was performed using FlowJo 10.8.0 software (FlowJo, LLC).

[0527] Bioluminescence-based cell survival in vitro assays

[0528] Cell lines (Jurkat, Nalm6, and OCLLyl8) were engineered to express CBG, and cell survival was measured using bioluminescent quantification. D-luciferin potassium salt (Gold Biotechnology; Cat# 115144-35-9) was added to cell cultures (final concentration 15 pg / mL) and incubated at 37°C for 10 min. Bioluminescent signal was detected using a BioTek Synergy H4 imager, and signal was analyzed using BioTek Gen5 software. Percent cytotoxicity was calculated using a control of target cells without effectors.

[0529] Flow cytometry-based cell survival in vitro assays

[0530] Primary T-ALL, AML, or Sezary cells were stained with CellTrace Violet (CTV; Invitrogen; Cat# C34557) prior to plating with CAR T-cells or control untransduced T-cells. Reagents were used according to the manufacturer's protocol. After 48 h, cells were stained with ViaKrome 808 Fixable Viability Dye (Beckman; Cat# C36628) and analyzed by flow cytometry (CytoFlex LX; Beckman) to determine the absolute count of ViaKrome 808-negative and CTV+cells. Absolute cell counts were determined using Flow-Count Fluorospheres (Beckman; Cat# 754053). Percent cytotoxicity was calculated using a control of target cells without effectors. Attorney Docket No. 046483 -7478WO 1(04048)

[0531] CellTrace Violet proliferation assays

[0532] T cells were resuspended in PBS containing CellTrace Violet (CTV; 1 : 1,000 dilution) at a concentration of 1 x 106cells per milliliter and incubated for 15 minutes at 37 °C. Cells were then washed with 10 mL of R10 medium, resuspended, and plated in co-culture with cancer cells at an effector-to-target (E:T) ratio of 0.25 : 1. After 5 days, proliferation was assessed by flow cytometry. The absolute number of CD3+T cells was determined using counting beads.

[0533] Mitochondrial Respiration (Agilent Seahorse XF)

[0534] Mitochondrial respiration was assessed using the Agilent Seahorse XF96 extracellular flux analyzer. XF96 wells were pre-coated with Cell-Tak (Corning) following the manufacturer’s instructions, incubated overnight at 37°C, air-dried, and stored at 4°C. Prior to assay, XF RPMI medium (non-buffered) supplemented with 10 mM glucose, 2 mM L-glutamine, and 5 mM HEPES was prepared fresh and adjusted to pH 7.4. T cells were centrifuged at 500 x g for 5 minutes, washed with PBS, and resuspended in XF assay medium. A total of 1 x 105cells per well were plated. The plate was centrifuged at 1,000 x g for 2 minutes and incubated at 37°C in a non-CC>2 incubator for 15 minutes. Baseline oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured, followed by sequential injections of 1.5 pM oligomycin A, 2.5 pM BAM15, and 0.5 pM rotenone with 0.5 pM antimycin A. ATP production rates were calculated using the Seahorse XF T Cell Metabolic Profiling Report Generator, which distinguishes mitochondrial ATP production (mitoATP, pmol ATP / min) from glycolytic ATP production (glycoATP, pmol ATP / min).

[0535] Cell-to-cell avidity experiments

[0536] Z-Movi-compatible acoustofluidic chips were coated with poly-L-lysine for 3h prior to attaching a monolayer of tumor cells. CellTrace Far-Red (Invitrogen; Cat# C34564) labelled CAR T-cells were allowed to incubate on the monolayer of tumor cells for 15 min, and then a ramping acoustic force was applied. Cell-detachment was analyzed using ImageJ and R. Avidity experiments were conducted according to the manufacturer's (LUMICKS™) instructions and recommendations.

[0537] Glass-supported planar lipid bilayer confocal imaging

[0538] Supported lipid bilayers were prepared by fusing small liposome droplets on clean glass coverslips as previously described (Guruprasad, et al., Nat Protoc. 2025 Jul 25, doi: 10.1038 / s41596-025-01208-x). Briefly, the liposome was trapped in a p-Slide VI 0.4 chamber Attorney Docket No. 046483 -7478WO 1(04048)

[0539] (Ibidi, Germany, Cat# 50-305-784). Lipid bilayers were first blocked with 5% casein for 20 min and then incubated with 6.3 nM of streptavidin (Thermo Fisher) for 15 min. After washing with imaging buffer (HEPES-buffered saline), lipids were incubated with biotinylated CD 19 protein (R&D Systems, Cat# 9269-CD) conjugated with AlexaFluor 488, respectively, at room temperature for 30 min. After washing with the imaging buffer, lipid bilayers were blocked with 2.5 |1M of D-biotin to saturate the streptavidin-binding sites. CAR T-cells were then stimulated on lipid bilayers for 30 min at 37°C. Cells were then fixed in 4% paraformaldehyde for 10 min and then permeabilized in 0.5% Triton-XlOO and 10% normal donkey serum in PBS for 30 min at room temperature. Then, the cells were stained with primary antibodies against CD2 (ThermoFisher, Cat# 914-MSM3-P1ABX) and phosphorylated CD3 zeta (Abeam, Cat# ab68236) or CD19 (R&D Systems, Cat# AVI9269). An Olympus FV3000 confocal microscope with 60x (NA 1.35) oil objective was used to obtain confocal image data. The fluorescence intensity is calculated as the sum of fluorescence at the synapse for individual cells.

[0540] Confocal imaging immune synapse with Leica Stellaris

[0541] Prior to the experiment, 18-well Ibidi chambers (81817, Ibidi) were covered with Poly-L lysine (P4707, Sigma) for 15mins at RT and washed 3X in PBS. Afterwards, 200 000 NALM6- GFP cells per well were seeded in RPMI supplemented with 2% FBS for 30 minutes to promote cell attachment to the chamber. Tumour cells were then co-cultured for 20 minutes with CD2 WT and KO CART 19 at a 1 : 1 ratio of CAR-positive to target cells. After the co-cultures, the samples were washed with PBS and fixed with 4% PFA (J19943.K2, ThermoFisher) for 15 minutes at room temperature. After fixation, the cells were washed 3x with PBS, blocked with PBS + 5% BSA (A7906, Sigma) for 30 mins at RT, and stained with mouse anti-human CD5 (UMAB9, ThermoFisher) at 1 / 100 dilution in blocking buffer for 1 hr at RT. After surface staining, samples were washed 3x with PBS and permeabilized with cold methanol for 10 mins at RT and blocked with PBS + 5% BSA for 30mins at RT. After blocking, samples were stained with rabbit anti-human pCD3z (ab68235, Abeam) overnight at 4°C. Then, the samples were washed 3x with PBS and stained with goat anti -mouse AF568 (Al 104, ThermoFisher) and goat anti-rabbit AF647 (A21245, ThermoFisher) both at 1 / 500 dilution in blocking buffer for 1 hr at RT. The samples were washed 3x with PBS and imaged on a Leica Stellaris with a 63x / 1.4 Oil objective. Attorney Docket No. 046483 -7478WO 1(04048)

[0542] In vivo immunodeficient mouse models

[0543] 6-10-week-old NOD-SCID-yc' ’ (NSG) mice were obtained from the Jackson Laboratory and maintained in pathogen-free conditions. All target cells were engineered to express CBG. Animals were injected via tail vein with IxlO6Jurkat, TH20, Nalm6, or Nalm6-PDL1- overexpressing cancer cells in 0.15 mb sterile PBS. For scRNAseq experiments, 5.0 x 106OCL Lyl8 were subcutaneously injected. After engraftment, IxlO6T-cells (CAR+or an equal number of UTD control) were injected via tail vein in 0.15 mb sterile PBS, unless otherwise indicated. Animals were monitored for signs of disease progression and overt toxicity, such as xenogeneic graft-versus-host disease, as evidenced by >20% loss in body weight, loss of fur, diarrhea, conjunctivitis, and disease-related hind limb paralysis. Disease burdens were monitored over time using a Xenogen IVIS bioluminescent imaging system or the Lumina S5 imaging system for tumor flux.

[0544] Single cell RNA sequencing and analysis

[0545] Peripheral blood was collected from all mice, followed by red blood cell lysis. Human cells were enriched using the EasySep Mouse / Human Chimera Isolation Kit (StemCell Technologies; cat. no. 19849) according to the manufacturer’s instructions. To further purify T cells, enriched samples were stained with anti-human CD3, CD5, and CD2 antibodies (APC, Biolegend) and sorted using a BD FACSMelody Cell Sorter. Prior to single-cell capture, cells were barcoded using TotalSeq-A Hashtagging Antibodies (BioLegend) to allow for sample multiplexing. After sorting, cells were washed twice with 0.04% BSA in PBS and loaded onto the 10X Chromium Controller. Libraries were prepared using the 10X Genomics v2 Chemistry kit, according to the manufacturer’s instructions. Next-generation sequencing on libraries was performed using the Illumina NovaSeq 6000. Raw single-cell RNA-seq data were processed using Cell Ranger software (v5.0.1; lOx Genomics), with transcript alignment and mapping performed against the GRC1138 human reference genome. Filtered gene expression data were processed in Seurat v 5.3.0 (Hao, et al., Cell. 2021 Jun 24;184(13):3573-3587.e29). Quality control filters were applied to retain cells with >200 and <5,500 detected RNA features and <10% mitochondrial gene content. Data were normalized and scaled using standard Seurat workflows. Dimensionality reduction was performed with principal component analysis by using a curated list of canonical T cell genes described by Szabo et al. to drive clustering (Szabo, et al., Nat Commun. 2019 Oct 17; 10(l):4706). The top 30 principal components were used to drive Attorney Docket No. 046483 -7478WO 1(04048) unsupervised clustering via UMAP at a resolution of 0.6. Differential gene expression between clusters across experimental groups was assessed using the FindMarkers function, which applies a two-sided Wilcoxon rank-sum test followed by Benjamini-Hochberg correction for multiple comparisons. For pathway analysis, we used clusterProfiler v4.0 (Wu, et al., clusterProfder 4.0: A universal enrichment tool for interpreting omics data. Innovation (Cambridge (Mass)). 2021 Aug 28;2(3): 100141).

[0546] CD2 patient validation cohorts

[0547] The flow cytometry archives (2002-2014) at the Children’s Hospital of Philadelphia (CHOP) were reviewed to identify cases of T- ALL in the pediatric and young adult age group (0- 25 years). Diagnosis of T-ALL was confirmed per WHO criteria (Arber, et al., Blood. 2016 May 19;127(20):2391-405). Archived flow cytometric histograms that were saved as PDFs were reviewed. Flow cytometric data were collected using Beckman Coulter FC 500 Series flow cytometers in a College of American Pathologists (CAP) accredited clinical flow cytometry laboratory. Bead controls, titration of newer antibody lots, and daily comparison of instruments were performed. One million cells / microliter single cell suspensions of bone marrow, peripheral blood or tissue were incubated with 5 color antibody panels including CD45-PE-Cy7, CD4-PE, CD8-FITC, CD7-PE-Cy5.5, CD3-ECD, CD2-FITC, CD56-PE, B-cell, myeloid markers, and isotype control antibodies (Beckman Coulter, CA). Twenty thousand events were collected. Data analysis was performed using FCS Express (De Novo software, CA). Negative gates were based on isotype controls. Immature blasts were identified using CD45 vs. SSC gating. T-cell markers CD2, sCD3, CD5, and CD7 were assessed on CD45dim blasts. Normal CD45bright mature T- cells served as controls for assessment of antigen.

[0548] The phenotypic profile of peripheral T-cell lymphomas from 91 patients diagnosed and treated at the European Institute of Oncology was analyzed via immunohistochemistry for CD2, CD3, CD5, and CD7 expression by a board-certified hematopathologist. CITE-seq data from patients enrolled in AALL0434 were obtained from Xu J, et al. (Tan, et al., Res Sq. 2023 Oct 30). AALL0434 (NCT00408005) was a Children’s Oncology Group (COG) phase 3 clinical trial that enrolled subjects from January 22, 2007 until July 25, 2014; trial results were previously published (Dunsmore, et al., J Clin Oncol. 2020 Oct l;38(28):3282-93). T-ALL subjects enrolled on a companion biology study used for sample banking, AALL03B1 (NCT00482352) from January 22, 2007 until August 08, 2010 or AALL08B1 (NCT01142427) from August 9, Attorney Docket No. 046483 -7478WO 1(04048)

[0549] 2010 until July 25, 2014. AALL03B1, AALL08B1 , and AALL0434 were approved by the NCI Cancer Evaluation and Therapeutic Program (CTEP), local IRBs at participating centers, and the Pediatric Central Institutional Review Board (IRB). Informed consent was obtained from all study participants and / or their legally authorized representative in accordance with the Declaration of Helsinki. Genomic studies were approved by the local IRB at the Children’s Hospital of Philadelphia, COG, and CTEP. Samples were decoded and assigned a unique study identifier (USI). Samples were banked at the COG biorepository at Nationwide Children’s Hospital in Chicago, IL. CITE-seq data were normalized using CLR normalization across cells (margin = 2). Original cell annotations, based on a 7-step process involving a projection to healthy pediatric thymus and bone marrow developmental reference, were retained. Data was integrated using the I tegrate Layers function in Seurat v5 using reciprocal PCA and visualized on a UMAP using the top 30 principal components (Hao, et al., Nat Biotechnol. 2024 Feb;42(2):293-304). Surface and RNA expression profiles were plotted using the FeaturePlot and VinPlot functions in Seurat v5 with default parameters. Surface and RNA expression for CD2 and other T-cell markers were plotted in n=16,199 healthy thymocytes from 3 pediatric donors and visualized using the VinPlot function in Seurat v5.

[0550] CD58 immunohistochemistry of primary B-cell lymphomas

[0551] CD58 immunohistochemical stain was performed on selected patient samples. Five- micron sections of formalin-fixed paraffin-embedded tissue were stained using antibody against CD58 at 1 : 100 dilution (R&D systems, Cat# AF1689). Staining was done on a Leica Bond- IIITM instrument using the Bond Polymer Refine Detection System (Leica Microsystems DS9800) and Goat IgG VisUCyte HRP Polymer (R&D Systems, Cat# VC004-25). Heat-induced epitope retrieval was done for 20 minutes with ER2 solution (Leica Microsystems AR9640). All the experiments were performed at room temperature. Slides were washed three times between each step with bond w...

Claims

Attorney Docket No. 046483 -7478WO 1(04048)CLAIMSWhat is claimed:

1. A chimeric switch receptor comprising:(i) an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain;(ii) a transmembrane domain; and(iii) an intracellular domain comprising a CD2 intracellular signaling domain.

2. The chimeric switch receptor of claim 1, wherein the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Immunoreceptor with Ig and ITEM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte-Associated Protein 4 (CTLA4); and wherein the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand-1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte- Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte- Associated Protein 4 (CTLA4).

3. The chimeric switch receptor of claim 1 or claim 2, wherein the transmembrane domain is a CD2 transmembrane domain.

4. The chimeric switch receptor of any one of the preceding claims, wherein the chimeric switch receptor comprises:(i) a PD1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain; or(ii) an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.Attorney Docket No. 046483 -7478WO 1(04048)5. The chimeric switch receptor of any one of the preceding claims, wherein the anticheckpoint inhibitor antigen binding domain is a single-chain variable fragment (scFv).

6. The chimeric switch receptor of any one of the preceding claims, wherein the chimeric switch receptor comprises an amino acid sequence having at least 80%, at least 81%, at least 82%>, at least 83%, at least 84%, at least 85%o, at least 86%, at least 87%o, at least 88%), at least 89%, at least 9O%o, at least 91%, at least 92%, at least 93%>, at least 94%, at least 95%>, at least 96%, at least 97%>, at least 98%>, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

7. The chimeric switch receptor of any one of the preceding claims, wherein the chimeric switch receptor comprises:(i) a leader sequence comprising an amino acid sequence having at least 80%, at least81%, at least 82%, at least 83%>, at least 84%, at least 85%o, at least 86%, at least87%, at least 88%, at least 89%>, at least 90%, at least 91%, at least 92%, at least93%o, at least 94%>, at least 95%>, at least 96%>, at least 97%o, at least 98%>, at least99%, or 100% sequence identity to SEQ ID NO: 2;(ii) a PD-1 LBD comprising an amino acid sequence having at least 80%, at least81%, at least 82%>, at least 83%>, at least 84%, at least 85%>, at least 86%, at least87%, at least 88%, at least 89%>, at least 90%, at least 91%, at least 92%, at least93%o, at least 94%, at least 95%>, at least 96%>, at least 97%>, at least 98%>, at least99%, or 100% sequence identity to SEQ ID NO: 3;(iii) a CD2 transmembrane domain comprising an amino acid sequence having at least80%o, at least 81%>, at least 82%>, at least 83%, at least 84%o, at least 85%>, at least86%, at least 87%, at least 88%>, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%>, at least 95%, at least 96%, at least 97%, at least98%o, at least 99%, or 100% sequence identity to SEQ ID NO: 4; and / or(iv) a CD2 intracellular signaling domain comprising an amino acid sequence having 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%,Attorney Docket No. 046483 -7478WO 1(04048) at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

8. A nucleic acid comprising a nucleotide sequence encoding the chimeric switch receptor of any one of the preceding claims.

9. A vector comprising the nucleic acid of claim 8.

10. The vector of claim 9, wherein the vector is a retroviral vector or a lentiviral vector.

11. A nucleic acid comprising:(i) a first nucleotide sequence encoding a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and(ii) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

12. The nucleic acid of claim 11, further comprising a self-cleaving peptide sequence between the first nucleotide sequence and the second nucleotide sequence, preferably wherein the self-cleaving peptide sequence is a P2A sequence.

13. The nucleic acid of claim 11 or claim 12, wherein the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte-Associated Protein 4 (CTLA4); andAttorney Docket No. 046483 -7478WO 1(04048) wherein the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand-1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte-Associated Protein 4 (CTLA4).

14. The nucleic acid of any one of claims 11-13, wherein the transmembrane domain of the chimeric switch receptor is a CD2 transmembrane domain.

15. The nucleic acid of any one of claims 11-14, wherein the chimeric switch receptor comprises:(i) a PD1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain; or(ii) an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

16. The nucleic acid of any one of claims 11-15, wherein the chimeric switch receptor comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 1.

17. The nucleic acid of any one of claims 11-16, wherein the chimeric switch receptor comprises:(i) a leader sequence comprising an amino acid sequence having at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to SEQ ID NO: 2;(ii) a PD-1 LBD comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at leastAttorney Docket No. 046483 -7478WO 1(04048)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 99%, or 100% sequence identity to SEQ ID NO: 3;(iii) a CD2 transmembrane domain comprising an amino acid sequence having at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; and / or(iv) a CD2 intracellular signaling domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 5.

18. The nucleic acid of any one of claims 11-17, wherein the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain.

19. The nucleic acid of any one of claims 11-18, wherein the CAR or the TCR targets a tumor antigen.

20. The nucleic acid of any one of claims 11-19, wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA- IX, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD 123, CD 133, CD 147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.Attorney Docket No. 046483 -7478WO 1(04048)21. The nucleic acid of any one of claims 11-20, wherein the anti -checkpoint inhibitor antigen binding domain of the chimeric switch receptor comprises a single-chain variable fragment (scFv).

22. The nucleic acid of any one of claims 11-21, wherein the tumor antigen binding domain of the CAR comprises a single-chain variable fragment (scFv).

23. The nucleic acid of any one of claims 11-22, wherein the intracellular domain of the CAR comprises a costimulatory domain of CD28, a costimulatory domain of 4- IBB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

24. The nucleic acid of any one of claims 11-23, wherein the CAR comprises an anti-CD19 antigen binding domain, a transmembrane domain, a 4- IBB costimulatory domain, and a CD3 zeta signaling domain.

25. A vector comprising the nucleic acid of any one of claims 11-24.

26. The vector of claim 25, wherein the vector is a retroviral vector or a lentiviral vector.

27. A modified immune cell, or precursor cell thereof, comprising the chimeric switch receptor of any one of claims 1-7, the nucleic acid of any one of claims 8 or 11-24, and / or the vector of any one of claims 9-10 or 25-26.

28. The modified immune cell or precursor cell thereof of claim 27, wherein the cell is a T cell, an autologous cell, a human cell, or any combination thereof.

29. A modified immune cell or precursor cell thereof, comprising:(i) a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, aAttorney Docket No. 046483 -7478WO 1(04048) transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and(ii) a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

30. The modified immune cell or precursor cell thereof of claim 29, wherein the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte- Associated Protein 4 (CTLA4); and wherein the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand-1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte-Associated Protein 4 (CTLA4).

31. The modified immune cell or precursor cell thereof of claim 29 or claim 30, wherein the transmembrane domain of the chimeric switch receptor is a CD2 transmembrane domain.

32. The modified immune cell or precursor cell thereof of any one of claims 29-31, wherein the chimeric switch receptor comprises:(i) a PD1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain; or(ii) an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

33. The modified immune cell or precursor cell thereof of any one of claims 29-32, wherein the chimeric switch receptor comprises an amino acid sequence having 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%, atAttorney Docket No. 046483 -7478WO 1(04048) least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

34. The modified immune cell or precursor cell thereof of any one of claims 29-33, wherein the chimeric switch receptor comprises:(i) a leader sequence comprising an amino acid sequence having at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to SEQ ID NO: 2;(ii) a PD-1 LBD comprising an amino acid sequence having at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to SEQ ID NO: 3;(iii) a CD2 transmembrane domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 4; and / or(iv) a CD2 intracellular signaling domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 5.

35. The modified immune cell or precursor cell thereof of any one of claims 29-34, wherein the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain.Attorney Docket No. 046483 -7478WO 1(04048)36. The modified immune cell or precursor cell thereof of any one of claims 29-35, wherein the CAR or the TCR targets a tumor antigen.

37. The modified immune cell or precursor cell thereof of any one of claims 29-36, wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA- A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD 19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, R0R1, R0R2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

38. The modified immune cell or precursor cell thereof of any one of claims 29-37, wherein the anti -checkpoint inhibitor antigen binding domain of the chimeric switch receptor comprises a single-chain variable fragment (scFv).

39. The modified immune cell or precursor cell thereof of any one of claims 29-38, wherein the tumor antigen binding domain of the CAR comprises a single-chain variable fragment (scFv).

40. The modified immune cell or precursor cell thereof of any one of claims 29-39, wherein the intracellular domain of the CAR comprises a costimulatory domain of CD28, a costimulatory domain of 4- IBB, an intracellular signaling domain of CD3 zeta, or any combination thereof.Attorney Docket No. 046483 -7478WO 1(04048)41 . The modified immune cell or precursor cell thereof of any one of claims 29-40, wherein the CAR comprises an anti-CD19 antigen binding domain, a transmembrane domain, a 4- 1BB costimulatory domain, and a CD3 zeta signaling domain.

42. The modified immune cell or precursor cell thereof of any one of claims 29-41, wherein the modified immune cell or precursor cell thereof comprises an expression vector comprising a first nucleotide sequence encoding the chimeric switch receptor and a second nucleotide sequence encoding the CAR or the TCR.

43. The modified immune cell or precursor cell thereof of claim 42, wherein the expression vector is a lentiviral vector or a retroviral vector.

44. The modified immune cell or precursor cell thereof of any one of claims 29-43, wherein the modified immune cell or precursor cell thereof is a T cell, an autologous cell, a human cell, or any combination thereof.

45. A pharmaceutical composition comprising a population of the modified immune cell or precursor cell thereof of any one of the preceding claims and a pharmaceutically acceptable carrier.

46. The pharmaceutical composition of claim 45, for use in a method of treating cancer in a subject in need thereof.

47. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of modified immune cells or precursor cells thereof, wherein the modified immune cells or precursor cells thereof comprise:(i) a chimeric switch receptor, wherein the chimeric switch receptor comprises an extracellular domain comprising a ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, aAttorney Docket No. 046483 -7478WO 1(04048) transmembrane domain, and an intracellular domain comprising a CD2 intracellular signaling domain; and(ii) a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR).

48. The method of claim 47, wherein the inhibitory immunoreceptor is selected from the group consisting of a Programmed Cell Death Protein 1 (PD1), a Transforming Growth Factor Beta Receptor I (TGFbRI), a Transforming Growth Factor Beta Receptor II (TGFbRII), a T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), a T Cell Immunoglobulin and Mucin Domain Containing 3 (TIM3), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte- Associated Protein 4 (CTLA4); and wherein the checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death Ligand- 1 (PD-L1), a Programmed Cell Death Protein 1 (PD1), a Lymphocyte-Activation Gene 3 (LAG3), and a Cytotoxic T-lymphocyte-Associated Protein 4 (CTLA4).

49. The method of claim 47 or claim 48, wherein the transmembrane domain of the chimeric switch receptor is a CD2 transmembrane domain.

50. The method of any one of claims 47-49, wherein the chimeric switch receptor comprises:(i) a PD1 LBD, a CD2 transmembrane domain, and a CD2 intracellular signaling domain; or(ii) an anti-PD-Ll antigen binding domain, a CD2 transmembrane domain, and a CD2 intracellular signaling domain.

51. The method of any one of claims 47-50, wherein the chimeric switch receptor comprises an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 1.

52. The method of any one of claims 47-51, wherein the chimeric switch receptor comprises:Attorney Docket No. 046483 -7478WO 1(04048)(i) a leader sequence comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 2;(ii) a PD-1 LBD comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 3;(iii) a CD2 transmembrane domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 4; and / or(iv) a CD2 intracellular signaling domain comprising an amino acid sequence having 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 99%, or 100% sequence identity to SEQ ID NO: 5.

53. The method of any one of claims 47-52, wherein the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain.

54. The method of any one of claims 47-53, wherein the CAR or the TCR targets a tumor antigen.

55. The method of any one of claims 47-54, wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276,Attorney Docket No. 046483 -7478WO 1(04048)CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIIT, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, Glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, R0R1, R0R2, TIM-3, TM4SF1, TnMucl, VEGFR2, and any combination thereof.

56. The method of any one of claims 47-55, wherein the anti-checkpoint inhibitor antigen binding domain of the chimeric switch receptor comprises a single-chain variable fragment (scFv).

57. The method of any one of claims 47-56, wherein the tumor antigen binding domain of the CAR comprises a single-chain variable fragment (scFv).

58. The method of any one of claims 47-57, wherein the intracellular domain of the CAR comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

59. The method of any one of claims 47-58, wherein the CAR comprises an anti-CD19 antigen binding domain, a transmembrane domain, a 4- IBB costimulatory domain, and a CD3 zeta signaling domain.

60. The method of any one of claims 47-59, wherein the modified immune cells or precursor cells thereof comprise an expression vector comprising a first nucleotide sequence encoding the chimeric switch receptor and a second nucleotide sequence encoding the CAR or the TCR.

61. The method of any one of claims 47-60, wherein the expression vector is a lentiviral vector or a retroviral vector.Attorney Docket No. 046483 -7478WO 1(04048)62. The method of any one of claims 47-61, wherein the modified immune cells or precursor cells thereof are T cells, autologous cells, human cells, or any combination thereof.

63. The method of any one of claims 47-62, wherein the subject is a human.

64. The method of any one of claims 47-63, wherein the cancer is selected from the group consisting of a B-cell malignancy (e.g., a B-cell lymphoma or a leukemia), lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophagus cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, and renal cancer.

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