Boosting chimeric antigen receptor cells in blood

Abstract

The present disclosure provides modified cells, i.e. immune cells or their precursor cells, wherein the cells are engineered to express (a) a first chimeric antigen receptor (CAR) that has affinity for CD19 and (b) a second CAR that has affinity for a tumor antigen that is not CD19. Also provided are methods and uses of modified cells, for example, for treating at least one sign and / or symptom of cancer in a subject. The modified cells are expanded in the peripheral blood of the subject. Also provided are related nucleic acids, vectors, and pharmaceutical compositions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 310,355, filed February 15, 2022, which is incorporated by reference herein in its entirety. [Background technology]

[0002] 2. Background of the Invention Chimeric antigen receptor T cell (CART) immunotherapy has shown remarkable improvements in clinical outcomes for patients with B-cell malignancies, including relapsed / refractory (r / r) lymphomas and leukemias (Lee, et al., The Lancet (2015) 385(9967):517-28 (Non-Patent Document 1); Maude et al., New England Journal of Medicine (2018) 378(5):439-48 (Non-Patent Document 2); Park et al., New England Journal of Medicine (2018) 378(5):449-59 (Non-Patent Document 3); Schuster, et al., New England Journal of Medicine (2019) 380(1):45-56 (Non-Patent Document 4); Turtle et al., Science Translational Medicine (2016) 8(355):355ra116-355ra116 (Non-Patent Document 5)). Despite the notable clinical results of anti-CD19 CART (CART19), the majority of patients treated with recently approved CART products fail these treatments. Analysis of acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL) patient samples from these trials revealed that the degree of response to CART19 therapy correlates with high levels of CAR T expansion in the blood and the acquisition and maintenance of B-cell aplasia. Unfortunately, patient responses to CAR T cell therapies designed to treat solid tumors are less effective. CAR T cells against solid tumor targets typically do not encounter their cognate targets in the blood, but instead undergo limited CAR T cell homeostatic expansion in the blood and are transported to the tumor site. The number of CAR T cells that reach the tumor is insufficient to eradicate the disease. Furthermore, increasing the infusion dose of CAR T cells has revealed on-target off-tumor toxicity to normal cells other than the tumor. There is a need in the art to enhance the anti-tumor efficacy of CAR adoptive cell therapy to improve patient clinical outcomes. The present invention addresses this need. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Lee, et al., The Lancet (2015) 385(9967):517-28 [Non-Patent Document 2] Maude et al., New England Journal of Medicine (2018) 378(5):439-48 [Non-Patent Document 3] Park et al., New England Journal of Medicine (2018) 378(5):449-59 [Non-Patent Document 4] Schuster, et al., New England Journal of Medicine (2019) 380(1):45-56 [Non-Patent Document 5] Turtle et al., Science Translational Medicine (2016) 8(355):355ra116-355ra116 Summary of the Invention

[0004] Disclosure Summary The present invention includes compositions and methods comprising modified cells, for example, for treating at least one sign and / or symptom of cancer in a subject. The modified cells are expanded in the peripheral blood of the subject. Related nucleic acids, vectors, and pharmaceutical compositions are also provided.

[0005] In such a case, in one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) a first extracellular antigen-binding domain having affinity for CD19; and (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and A first nucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising: b. (i) a second extracellular antigen-binding domain having affinity for a tumor antigen that is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and A second nucleotide sequence encoding a second CAR comprising: The present invention provides an isolated nucleic acid comprising:

[0006] In one embodiment, the first nucleotide sequence is located 5' to the second nucleotide sequence.

[0007] In certain embodiments, the first nucleotide sequence is located 3' to the second nucleotide sequence.

[0008] In one embodiment, the isolated nucleic acid further comprises a linker nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, wherein the linker nucleotide sequence comprises a ribosome slippage sequence selected from the group consisting of P2A, T2A, E2A, and F2A.

[0009] In certain embodiments, the isolated nucleic acid further comprises a promoter operably linked to the first nucleotide sequence and / or the second nucleotide sequence.

[0010] In one embodiment, the promoter is an EF1α promoter.

[0011] In certain aspects, the isolated nucleic acid further comprises a post-transcriptional regulatory element.

[0012] In one embodiment, the post-transcriptional regulatory element is a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE).

[0013] In one embodiment, the tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, 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, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFRα4); HER2, HLA-A2, ICAM1, interleukin-13 receptor subunit alpha (IL3Rα), interleukin-13 receptor subunit alpha 1 (IL13Rα1), interleukin-13 receptor subunit alpha 2 (IL13Rα2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC 1, Nectin4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1-6), New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1), P16, PD-L1, Prostate Stem Cell Antigen (PSCA), Prostate Specific Membrane Antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), VEGFR2, and any combination thereof.

[0014] In certain embodiments, the tumor antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFRalpha4), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2).

[0015] In certain embodiments, the first extracellular antigen binding domain and the second extracellular antigen binding domain are independently selected from a single chain variable fragment (scFv) and a Fab, respectively.

[0016] In certain embodiments, at least one costimulatory domain of the first or second intracellular domain comprises a costimulatory domain of a protein independently selected from 4-1BB, CD2, CD28, and ICOS.

[0017] In one embodiment, the signaling domain comprises a CD3 zeta signaling domain.

[0018] In certain embodiments, the first and / or second transmembrane domain comprises a transmembrane domain of a protein independently selected from CD8, CD28, and 4-1BB.

[0019] In some embodiments, the first and / or second CAR further comprises a hinge domain.

[0020] In one embodiment, the hinge domain comprises a CD8 hinge domain.

[0021] In one aspect, the invention provides an isolated nucleic acid as disclosed herein, comprising: a. The first CAR is: (i) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Includes; b. The second CAR is: (i) a second extracellular antigen-binding domain comprising an anti-tumor antigen scFv, wherein the tumor antigen is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Includes.

[0022] In one aspect, the invention provides an isolated nucleic acid as disclosed herein, comprising: a. The first CAR is: (i) the CD8 leader sequence; (ii) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; and (iii) CD8 hinge and; (iv) a CD28 transmembrane domain; and (v) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and consists essentially of; b. The second CAR is: (i) the CD8 leader sequence; (ii) a second extracellular antigen-binding domain comprising an anti-mesothelin scFv; and (iii) CD8 hinge and; (iv) a CD8 transmembrane domain; and (v) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and It essentially consists of:

[0023] In an embodiment, the isolated nucleic acid further comprises a third nucleotide sequence encoding a switch receptor, wherein the switch receptor comprises an extracellular domain of a first receptor and an intracellular domain of a second receptor, the first receptor and the second receptor being, respectively, TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28. , TIM3 and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, VISTA and IL-9R, TIGIT and IL-9R, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R.

[0024] In one embodiment, the isolated nucleic acid further comprises a fourth nucleotide sequence encoding a dominant negative receptor that is a dnTGFβR.

[0025] In one aspect, the invention provides a vector comprising an isolated nucleic acid disclosed herein.

[0026] In certain aspects, the vector is a lentiviral vector or a retroviral vector.

[0027] In one aspect, the invention provides modified cells comprising an isolated nucleic acid disclosed herein or a vector disclosed herein.

[0028] In certain aspects, the cell is selected from a bacterial cell, a fungal cell, a yeast cell, an insect cell, an animal cell, a mammalian cell, and a human cell.

[0029] In certain aspects, the cell is a mammalian cell or a human cell, and the cell is an immune cell or a precursor thereof.

[0030] In some embodiments, the immune cell is a T cell.

[0031] In one aspect, the cell is an immune cell or a precursor thereof, the cell being (i) a first extracellular antigen-binding domain having affinity for CD19; and (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and A first chimeric antigen receptor (CAR) comprising: b. (i) a second extracellular antigen-binding domain having affinity for a tumor antigen that is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and The second CAR containing It has been engineered to express

[0032] In one embodiment, the tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, 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, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin-13 receptor subunit alpha (IL3Rα), interleukin-1 3 receptor subunit alpha 1 (IL13Rα1), interleukin 13 receptor subunit alpha 2 (IL13Rα2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin-4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1–6), New York squamous esophageal The IL-16 receptor agonist and / or IL-16 receptor agonist are selected from the group consisting of: epidermal growth factor stimulatory system (EMS)-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4; GFR alpha 4), VEGFR2, and any combination thereof.

[0033] In certain embodiments, the tumor antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), glycosyl-phosphatidylinositol (GPI)-linked GDNF family alpha-receptor 4 (GFRα4; GFRalpha4), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), and interleukin-13 receptor subunit alpha 2 (IL13Rα2).

[0034] In certain embodiments, the first extracellular antigen binding domain and the second extracellular antigen binding domain are independently selected from a single chain variable fragment (scFv) and a Fab, respectively.

[0035] In certain embodiments, at least one costimulatory domain of the first or second intracellular domain comprises a costimulatory domain of a protein independently selected from 4-1BB, CD2, CD28, and ICOS.

[0036] In one embodiment, the signaling domain comprises a CD3 zeta signaling domain.

[0037] In certain embodiments, the first and / or second transmembrane domain comprises a transmembrane domain of a protein independently selected from CD8, CD28, and 4-1BB.

[0038] In some embodiments, the first and / or second CAR further comprises a hinge domain.

[0039] In one embodiment, the hinge domain comprises a CD8 hinge domain.

[0040] In one aspect, the invention provides a modified cell as disclosed herein, comprising: a. The first CAR is: (i) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Includes; b. The second CAR is: (i) a second extracellular antigen-binding domain comprising an anti-tumor antigen scFv, wherein the tumor antigen is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Includes.

[0041] In one aspect, the invention provides a modified cell as disclosed herein, comprising: a. The first CAR is: (i) the CD8 leader sequence; (ii) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; and (iii) CD8 hinge and; (iv) a CD28 transmembrane domain; and (v) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and consists essentially of; b. The second CAR is: (i) the CD8 leader sequence; (ii) a second extracellular antigen-binding domain comprising an anti-mesothelin scFv; and (iii) CD8 hinge and; (iv) a CD8 transmembrane domain; and (v) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and It essentially consists of:

[0042] In some embodiments, the cell is further engineered to express a switch receptor, wherein the switch receptor comprises an extracellular domain of a first receptor and an intracellular domain of a second receptor, the first receptor and the second receptor being, respectively, TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, TIM3 and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, VISTA and IL-9R, TIGIT and IL-9R, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R.

[0043] In one embodiment, the cells are further engineered to express a dominant negative receptor that is a dnTGFβR.

[0044] In one embodiment, the cell comprises a vector encoding a first CAR and a second CAR.

[0045] In certain aspects, the vector is a lentiviral vector or a retroviral vector.

[0046] In certain embodiments, the vector further encodes a switch receptor as disclosed herein and / or a dominant negative receptor as disclosed herein.

[0047] In certain embodiments, the cell is a mouse cell or a human cell.

[0048] In some embodiments, the cell is a T cell.

[0049] In one aspect, the present invention provides pharmaceutical compositions comprising a population of modified cells disclosed herein and at least one pharma- ceutically acceptable carrier.

[0050] In one aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition disclosed herein.

[0051] 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 population of modified cells, wherein the cells are immune cells or precursor cells thereof, (i) a first extracellular antigen-binding domain having affinity for CD19; and (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and A first chimeric antigen receptor (CAR) comprising: b. (i) a second extracellular antigen-binding domain having affinity for a tumor antigen that is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and The second CAR containing It has been engineered to express

[0052] In some embodiments, the tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, Claude IL-18.2, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin-13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin 4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1–6), New York esophageal squamous cell carcinoma-1 (NY-E SO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4; GFR alpha 4), VEGFR2, and any combination thereof.

[0053] In certain embodiments, the tumor antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), glycosyl-phosphatidylinositol (GPI)-linked GDNF family alpha-receptor 4 (GFRα4; GFRalpha4), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2).

[0054] In certain embodiments, the first extracellular antigen binding domain and the second extracellular antigen binding domain are independently selected from a single chain variable fragment (scFv) and a Fab, respectively.

[0055] In certain embodiments, at least one costimulatory domain of the first or second intracellular domain comprises a costimulatory domain of a protein independently selected from 4-1BB, CD2, CD28, and ICOS.

[0056] In one embodiment, the signaling domain comprises a CD3 zeta signaling domain.

[0057] In certain embodiments, the first and / or second transmembrane domain comprises a transmembrane domain of a protein independently selected from CD8, CD28, and 4-1BB.

[0058] In some embodiments, the first and / or second CAR further comprises a hinge domain.

[0059] In one embodiment, the hinge domain comprises a CD8 hinge domain.

[0060] In one aspect, the present invention provides a method as disclosed herein, comprising: a. The first CAR is: (i) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Includes; b. The second CAR is: (i) a second extracellular antigen-binding domain comprising an anti-tumor antigen scFv, wherein the tumor antigen is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Includes.

[0061] In one aspect, the invention provides a method as disclosed herein, comprising: a. The first CAR is: (i) the CD8 leader sequence; (ii) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; and (iii) CD8 hinge and; (iv) a CD28 transmembrane domain; and (v) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and consists essentially of; b. The second CAR is: (i) the CD8 leader sequence; (ii) a second extracellular antigen-binding domain comprising an anti-mesothelin scFv; and (iii) CD8 hinge and; (iv) a CD8 transmembrane domain; and (v) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and It essentially consists of:

[0062] In some embodiments, the modified cell is further engineered to express a switch receptor, wherein the switch receptor comprises an extracellular domain of a first receptor and an intracellular domain of a second receptor, the first receptor and the second receptor being, respectively, TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, TIM 3 and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, VISTA and IL-9R, TIGIT and IL-9R, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R.

[0063] In one embodiment, the modified cells are further engineered to express a dominant negative receptor that is a dnTGFβR.

[0064] In some embodiments, the modified cell comprises a vector encoding a first CAR and a second CAR.

[0065] In certain aspects, the vector is a lentiviral vector or a retroviral vector.

[0066] In one embodiment, the vector further encodes a switch receptor according to embodiment 57 and / or a dominant negative receptor according to embodiment 58.

[0067] In some aspects, the modified cell is a mouse cell or a human cell.

[0068] In some embodiments, the population of modified cells comprises T cells.

[0069] In some aspects, the modified cells are autologous to the subject.

[0070] In some aspects, the modified cells are allogeneic to the subject.

[0071] In certain embodiments, the population of modified cells is administered as a pharmaceutical composition comprising the population of modified cells and at least one pharma- ceutically acceptable carrier.

[0072] In one embodiment, the population of modified cells is about 1×10 6 ~Approx. 1×10 9 Contains cells.

[0073] In some aspects, the modified cells exhibit expansion in the peripheral blood of the subject.

[0074] In certain embodiments, the expansion is at least 10-fold, at least 100-fold, or at least 1000-fold.

[0075] In some embodiments, the modified cells are detectable for at least 24 months after administration of the cells.

[0076] In some embodiments, the subject is a human.

[0077] In certain embodiments, the cancer is selected from breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, prostate cancer, pancreatic cancer, colon cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid cancer.

[0078] In certain embodiments, the population of modified cells comprises T cells, and at least 30% or at least 40% of the population of modified cells at or after day 7 post-administration are phenotypically central memory T cells.

[0079] In certain embodiments, the method further comprises administering a CD19 antigen to the subject.

[0080] In certain embodiments, the step of administering the CD19 antigen comprises administering a vector encoding the CD19 antigen or cells engineered to express the CD19 antigen.

[0081] In certain embodiments, the CD19 antigen comprises the CD19 extracellular domain or an antigenic fragment thereof.

[0082] In certain embodiments, the CD19 antigen is administered prior to, simultaneously with, or following administration of the population of modified cells.

[0083] In one embodiment, the vector encoding the CD19 antigen is an adenoviral vector.

[0084] In certain embodiments, the method further comprises administering an anti-PD1 immunotherapy to the subject.

[0085] In some embodiments, the anti-PD1 immunotherapy is an anti-PD1 antibody. [Brief description of the drawings]

[0086] 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.

[0087] [Figure 1A]Figure 1A-1B show the design and expression of two pTRPE lentiviral constructs for expression of dual boost CARs in the same cells. One CAR contains an anti-human CD19 scFv, a CD28 costimulatory domain and a CD3 zeta signaling domain (hCD19 / 28z) to boost CAR T cells in peripheral blood (PB). The other CAR contains an scFv targeting a tumor antigen (an example here is M5, an anti-human mesothelin (MSLN) scFv) and further contains a 4-1BB costimulatory domain and a CD3 zeta signaling domain (M5BBZ). Figure 1A provides a schematic of the pTRPE-hCD19 / 28z-M5BBZ and pTRPE-M5BBZ-hCD19 / 28z dual CAR constructs. Figure 1B provides flow cytometry data showing expression of hCD19 / 28z and M5BBZ CARs in T cells from lentiviral constructs. [Figure 1B] See legend to Figure 1A. [Figure 2A] Figures 2A-B provide data showing the expansion of dual CAR T cells when co-cultured in vitro with irradiated Nalm6 human tumor cell line (expressing CD19). Figure 2A provides a graph showing viable cell numbers after 4 days of co-culture with irradiated CD19+ Nalm6 for T cells transduced with the indicated constructs compared to the absence of irradiated Nalm6. UTD, untransduced. Figure 2B provides CSFE staining data showing that CAR T expansion in CD4+ and CD8+ cells is similar for T cells transduced with either dual CAR constructs (pTRPE-M5BBZ-hCD19 / 28z or pTRPE-hCD19 / 28z-M5BBz) or single anti-CD19 CAR construct (pNVS-hCD19BBZ), but not for T cells transduced with a single M5 CAR or untransduced (UTD) T cells. [Figure 2B-1] See legend to Figure 2A. [Figure 2B-2] See legend to Figure 2A. [Figure 2B-3] See legend to Figure 2A. [Figure 2B-4] See legend to Figure 2A. [Figure 2B-5] See legend to Figure 2A. [Figure 3A] Figures 3A-B provide data on the in vitro cytotoxicity of CAR T cells against mesothelin-positive pancreatic tumor AsPC1 cells. Figure 3A shows that T cells transduced with pTRPE-hCD19 / 28z-M5BBZ or pTRPE-M5BBZ-hCD19 / 28z constructs kill AsPC1 cells. Figure 3B shows that co-culture of dual CART cells with irradiated Nalm6 cells promotes the killing of AsPC1 cells by dual CART cells. Enhanced killing of AsPC1 cells was higher for T cells transduced with pTRPE-hCD19 / 28z-M5BBZ construct compared to T cells transduced with pTRPE-M5BBZ-hCD19 / 28z. [Figure 3B] See legend to Figure 3A. [Figure 4A]Figures 4A-4D provide data on the cytotoxicity of CAR T cells against type 2 pneumocyte-derived mesothelin-positive A549 cells, which serve as a model for measuring potential toxicity against non-tumor normal cells in lung tissue. Figures 4A and 4B provide graphs showing the results that T cells containing the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct exhibit reduced cytotoxicity of A549 cells compared to T cells transduced with the pTRPE-M5BBZ single CAR construct or the pTRPE-M5BBZ-hCD19 / 28z dual CAR construct, respectively. Figure 4C shows the results that T cells transduced with the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct exhibit reduced cytotoxicity of A549 cells compared to T cells containing the pTRPE-M5BBZ-hCD19 / 28z dual CAR construct after co-culture with irradiated Nalm6 cells. Figure 4D provides data showing the results that T cells transduced with the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct express lower levels of the M5BBZ CAR compared to T cells transduced with the pTRPE-M5BBZ-hCD19 / 28z dual CAR construct. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D-1] See legend to Figure 4A. [Figure 4D-2] See legend to Figure 4A. [Figure 5A]Figures 5A-5F provide data showing the results that irradiated NALM6 stimulates the expansion of dual CAR T cells in vivo in tumor-free NSG mice. Figure 5A is a schematic of the experiment. Figure 5B provides data showing the in vivo expansion of T cells transduced with either the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct or the pTRPE-M5BBZ-hCD19 / 28z dual CAR construct. Figure 5C provides data showing that T cells transduced with the pTRPE-M5BBZ construct did not expand. Figure 5D provides data showing the expansion of dual CAR T in various organs. Figure 5E provides data showing a significant increase in the secretion of cytotoxic cytokines (interferon production regulator (IFNr), perforin, granzyme A, and granulysin) in serum. Figure 5F provides data regarding the phenotype of T cells recovered at day 20. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 5F-1] See legend to Figure 5A. [Figure 5F-2] See legend to Figure 5A. [Figure 5F-3] See legend to Figure 5A. [Figure 6A]Figures 6A-6D provide data showing the results that irradiated NALM6 enhances dual CAR T cell killing in vivo of AsPC1 pancreatic tumors in NSG mice. Figure 6A is a schematic of the experiment for mice that did not receive irradiated Nalm6. Figure 6B provides a graph plotting tumor size versus days after T cell injection showing the results that T cells transduced with pTRPE-hCD19 / 28z-M5BBZ dual CAR constructs show significantly enhanced killing of pancreatic tumors compared to T cells transduced with pTRPE-M5BBZ single CAR constructs. Figure 6C provides a graph plotting tumor size versus days after T cell injection showing the results that irradiated NALM6 enhances and promotes dual CAR T cell killing in vivo of AsPC1 pancreatic tumors in NSG mice. Figure 6D provides data showing that enhanced in vivo killing of pancreatic tumors for T cells transduced with the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct compared to the pTRPE-M5BBZ single CAR construct correlates with higher CAR T numbers in the tumor for T cells transduced with the dual CAR construct and transition of CAR T cells into CD4+ cells. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 7A]Figures 7A-7E provide data on the cytotoxicity of murine dual CARs in syngeneic mice. Figure 7A provides a graph showing in vitro CAR T cell killing of the mesothelin-positive PDA7940bWT cell line for murine T cells transduced with the indicated constructs. Figure 7B provides data showing a decrease in CD19+ cells and an increase in CD45.1+ cells in mice receiving murine T cells transduced with the murine dual CAR constructs but not either single CAR construct without lymphocyte depletion. Figure 7C provides data showing that expanded dual CAR T cells are primarily in the spleen compared to the liver. Figure 7D provides data on the phenotype of T cells in the blood collected on day 7. Figure 7E provides data on the phenotype of T cells in the spleen collected on day 7. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D-1] See legend to Figure 7A. [Figure 7D-2] See legend to Figure 7A. [Figure 7E-1] See legend to Figure 7A. [Figure 7E-2] See legend to Figure 7A. [Figure 8] Schematic of the experimental design. C57BL / 6 syngeneic mice were implanted with PDA (pancreatic) tumors on day -7. Mice were injected with 1e6 CD45.1+ CAR T cells (transduced with MSGV-anti-moCD19-MuCD28z-anti-mo meso-A03-3-MuBBz dual CAR construct or MSGV-anti-mo meso-A03-3-MuBBz single CAR construct) on day 0. Mice then received CD19 antigen (as 1e9 pfu of Ad-CMV-mCD19t-P2A-eGFP) intratumorally every other day from day 5-11. [Figure 9] 1 provides data showing expression of CD45.1 by flow cytometry on tumor samples 11 days after CAR-T infusion. [Figure 10]1 provides data showing CD45.1 expression by flow cytometry on tumor samples 11 days after CAR-T infusion. Null refers to the PBS control. [Figure 11] 1 provides data showing PD-1 expression on CD8+CD45.1+ cells by flow cytometry on tumor samples 11 days after CAR-T infusion. [Figure 12] 1 provides a graph plotting tumor size versus days after CAR-T infusion. [Figure 13A] Figures 13A-13B provide data showing expression of CD45.1 (Figure 13A) and PD-1 (Figure 13B) on CD8+CD45.1+ cells by flow cytometry on tumor samples 11 days after CAR-T infusion. [Figure 13B] See legend to Figure 13A. [Figure 14A] Figures 14A-14D provide data on the results that dual-targeted CAR T cells show increased tumor burden control and overall survival in vivo in "hot" PDA tumors. Figure 14A is a schematic of the experimental design. mCAR T cells were injected 7 days after engraftment of "hot" PDA tumors. Mice were treated intermittently with adenovirus (AAV) encoding truncated CD19. Figure 14B is a graph showing tumor burden (n=10 / group, N=2). P values ​​were determined using repeated measures two-way ANOVA with Tukey's multiple comparison test. Compared to A03, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Compared to A03-AAV, +p<0.05, ++p<0.01, +++p<0.001, ++++p<0.0001. Figure 14C is a graph showing overall survival for mice not administered AAV. P values ​​were determined by log-rank (Mantel-Cox) test. Figure 14D is a graph showing overall survival for mice administered AAV. P values ​​were determined by log-rank (Mantel-Cox) test. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 15A] Figures 15A-15B provide data on the results that dual-targeted CAR T cells have increased proliferation and effector phenotype in the "cold" PDA model and induce B cell aplasia in the spleen. The experimental scheme shown in Figure 14A was adapted for use with the "cold" tumor PDA model. CD45.1+ CAR T cells from the spleen were characterized 6 days after CAR T infusion via bulk RNA-seq (n=5 / replicate). Figure 15A is a plot of the top differentially expressed genes from data normalized and analyzed using DESeq2. Figure 15B is a chart showing gene set enrichment analysis prepared using the fGSEA package. [Figure 15B] See legend to Figure 15A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] Detailed Description Although CAR-T cell therapy has been successful in treating hematological malignancies, it is not yet effective in treating solid tumors. There are important differences between treating liquid and solid tumors. Intravenously administered CAR T cells must be transported throughout the body to find solid tumor sites compared to hematological malignancies. T cell trafficking is limited in solid tumors, as is the case in brain tumors due to the blood-brain barrier. Moreover, tumor heterogeneity is a fundamental property of cancer. In such cases, targeting one antigen (Ag) results in relapse associated with the reduction of that Ag induced by CAR T cell therapy. Meanwhile, CAR-T cells can be rendered dysfunctional by both intrinsic and extrinsic factors. Intrinsic dysfunction is mainly due to T cell exhaustion driven by high antigen load and / or persistent signaling. The function of CAR T cells can also be inhibited by exogenous factors, such as cytokines (e.g., TGF-β), ligands signaling through inhibitory receptors (e.g., PD-L1), or nutrient competition within the tumor microenvironment (TME). Thus, poor engraftment and limited expansion of CAR T cells are two major reasons why CAR T therapy fails to eliminate solid tumors.

[0089] Furthermore, the typical therapeutic dose of CAR T cells in human patients is approximately 10 7 ~10 9 cells, whereas typical therapeutic doses of CAR T cells used in preclinical mouse models range from 10 6 ~10 7 cell range, which is 10 10 ~10 11cells. However, simply increasing the therapeutic dose of CAR T cells administered to human patients is known to increase the risk of toxicity to normal cells outside of the tumor. For example, a recent clinical trial treated mesothelin (MSLN)-positive ovarian and pancreatic cancer patients with T cells expressing a CAR containing anti-MSLN scFv ("M5"). Expression of MSLN was high in the patients' tumors. However, MSLN was also expressed at low levels in some normal tissues, such as type 2 pneumocytes in the lung. Results from this trial showed that a low dose (3 × 10 7 M5 CAR-T cells at a dose of 1–3 × 10 were well tolerated by patients but did not provide antitumor efficacy, whereas higher doses (1–3 × 10 8 showed that administration of 1000 M5 CAR-T cells resulted in severe respiratory problems (potentially due to toxicity to non-tumor normal cells) and yet had no therapeutic effect. Although local administration of CAR-T cells at the tumor site is possible, this strategy limits the therapeutic potential for metastatic cancer. Overall, this trial showed that low doses of CAR-T cells typically fail to eliminate cancer, whereas higher doses of CAR-T cells cause toxicity to non-tumor normal cells and have no therapeutic effect. Thus, there is a need for strategies to deliver therapeutically effective doses of CAR-T cells to solid tumors while simultaneously reducing toxicity.

[0090] The present invention involves the use of CD19 antigen-driven expansion of CAR-expressing immune cells (e.g., CAR T cells) in the peripheral blood (PB) of a patient to safely increase the therapeutic index of adoptive cell therapy targeting solid tumors. This is achieved using a dual CAR approach, in which a subject receives immune cells (i.e., multiple immune cells, e.g., multiple T cells) engineered to express a first CAR that targets CD19 antigen and a second CAR that targets a tumor antigen other than CD19 (i.e., dual CAR T cells; dual CAR immune cells). The CD19 antigen in the subject drives the expansion of the dual CAR T cells. The CD19 antigen is present endogenously to the subject, e.g., on CD19-expressing B cells, and / or is provided exogenously, e.g., as a CD19 antigen protein, as a cell expressing the CD19 antigen, or as a nucleic acid comprising a nucleotide sequence encoding the CD19 antigen. Compared to cells expressing a single CAR targeting a tumor antigen, the dual CAR T cell approach of the present invention surprisingly enhances the in vivo expansion of CAR T cells in both peripheral blood and organs of subjects, enhances and promotes tumor cell killing (e.g., solid tumor cell killing and tumor size reduction) in vitro and in vivo, increases the expression of cytotoxic cytokines, and reduces cytotoxicity against non-tumor normal cells. Importantly, the boost and anti-tumor efficacy do not require lymphodepletion. Additionally, the boosted dual CAR T cells of the present invention are predominantly effector memory or central memory phenotypes and express only one or two exhaustion markers. Importantly, these unexpected effects are not simply additive effects of combining two CARs, and the present invention addresses a long-standing unmet need to treat solid tumors with adoptive cell therapy.

[0091] In one aspect, the invention provides an isolated nucleic acid comprising: (a) a first nucleotide sequence encoding a first chimeric antigen receptor (CAR), the first nucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second nucleotide sequence encoding a second CAR, the second nucleotide sequence encoding a second chimeric antigen receptor (CAR) comprising (i) a second extracellular antigen binding domain having affinity for a tumor antigen that is not CD19, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain.

[0092] In another aspect, the invention provides modified cells, wherein the cell is an immune cell or a precursor thereof, and the cell has been engineered to express (a) a first chimeric antigen receptor (CAR) comprising (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second CAR comprising (i) a second extracellular antigen binding domain having affinity for a tumor antigen that is not CD19, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain.

[0093] 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 population of modified cells, wherein the cells are immune cells or precursors thereof, and the cells are engineered to express: (a) a first chimeric antigen receptor (CAR) comprising: (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second CAR comprising: (i) a second extracellular antigen binding domain having affinity for a tumor antigen, where the tumor antigen is not CD19, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain.

[0094] In other aspects, provided herein are related vectors, compositions (eg, pharmaceutical compositions), and kits.

[0095] It is to be understood that the methods described in this disclosure are not limited to the particular methods and experimental conditions disclosed herein, and that 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.

[0096] Furthermore, the experiments described herein use, unless otherwise indicated, conventional molecular and cell biological and immunological techniques within the skill of the art.Such techniques are well known to those skilled in the art and are fully described in the literature.See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (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).

[0097] Methods and techniques using T cells bearing chimeric antigen receptors (CAR T cells) are described, for example, in 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 incorporated by reference herein in their entireties.

[0098] A. Definition Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of any potential meaning ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Unless otherwise stated, the use of "or" means "and / or". The use of the term "including" and other forms such as "includes" and "included" are non-limiting.

[0099] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and widely used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art, unless otherwise indicated, and as described in various general and more specific references cited and discussed throughout the specification. Enzymatic reactions and purification techniques are generally performed according to manufacturer's specifications as generally accomplished in the art, or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, and the experimental procedures and techniques thereof, are well known and widely used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0100] In order that this disclosure may be more readily understood, selected terms are defined below.

[0101] 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.

[0102] "About" when used herein when referring to a measurable value such as amount, duration, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the stated value, where such variations are appropriate for performing the disclosed methods.

[0103] "Activation" as used herein refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, among other things, to T cells undergoing cell division.

[0104] As used herein, "alleviating" a disease means reducing the severity of one or more symptoms of the disease.

[0105] The term "antigen" as used herein is defined as a molecule that elicits an immune response. This immune response may involve either or both of antibody production or activation of specific immunologically competent cells. Those skilled in the art will appreciate that any macromolecule can serve as an antigen, including virtually any protein or peptide.

[0106] Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art understand that any DNA that contains a nucleotide sequence or partial nucleotide sequence that encodes a protein that induces an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art understand that an antigen does not have to be encoded only by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to induce a desired immune response. Moreover, those skilled in the art understand that an antigen does not have to be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0107] The term "autologous" as used herein is meant to refer to any material derived from the same individual that is later reintroduced into the individual.

[0108] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0109] A "costimulatory signal" as used herein refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in T cell proliferation and / or up- or down-regulation of key molecules.

[0110] A "disease" is an animal's health condition in which the animal is unable to maintain homeostasis and the animal's health will continue to deteriorate unless the disease is improved.In contrast, an "injury" in an animal is a health condition in which the animal is able to maintain homeostasis, but the animal's health condition is less favorable than in the absence of the disorder.If left untreated, the disorder does not necessarily cause further deterioration in the animal's health condition.

[0111] The term "downregulation" as used herein refers to a decrease or elimination of gene expression of one or more genes.

[0112] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance, or composition described herein that is effective to achieve a particular biological result or provide 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 an immune response detected in the absence of the composition of the present invention. Immune response can be easily assessed by numerous methods recognized in the art. Those skilled in the art will understand that the amount of the composition administered herein will vary and can be easily 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 others.

[0113] "Encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a predetermined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a predetermined amino acid sequence and the biological properties attributed thereto. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other living system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0114] "Endogenous" as used herein refers to any substance produced from or within an organism, cell, tissue or system.

[0115] The term "epitope" as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response that induces a B and / or T cell response. An antigen can have one or more epitopes. Most antigens have multiple epitopes; i.e., they are multivalent. Generally, epitopes are approximately about 10 amino acids and / or sugars in size. Preferably, epitopes are about 4-18 amino acids, more preferably about 5-16 amino acids, even more preferably about 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. Those skilled in the art will appreciate that generally, the overall three-dimensional structure of the molecule, rather than the specific linear sequence, is the primary criterion for antigen specificity and thus distinguishes epitopes from one another. Based on the present disclosure, the peptides used in the present invention can be epitopes.

[0116] The term "exogenous" as used herein refers to any substance introduced from or produced outside an organism, cell, tissue or system.

[0117] The term "expand" as used herein refers to increasing in number, such as increasing the number of T cells. In one embodiment, T cells expanded ex vivo are increased in number compared to the number naturally present in the culture. In another embodiment, T cells expanded ex vivo are increased in number compared to other cell types in the culture. The term "ex vivo" as used herein refers to cells removed from a living organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0118] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0119] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence that is operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art that incorporate recombinant polynucleotides, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0120] "Identity" as used herein refers to the identity of subunit sequences between two polymers, particularly between two amino acid molecules, for example, between two polypeptide molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer 10 amino acids long) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, the two amino acid sequences are 90% identical.

[0121] The term "immune response" as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify the antigenic molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0122] The term "immunosuppressive" is used herein to refer to reducing the overall immune response.

[0123] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as, for example, a host cell.

[0124] "Lentivirus" as used herein refers to a genus of the Retroviridae family.Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information to the DNA of host cells, making them one of the most efficient methods of gene delivery vectors.HIV, SIV, and FIV are all examples of lentiviruses.Vectors derived from lentiviruses provide a means to achieve significant levels of in vivo gene transfer.

[0125] By the term "modified" as used herein is meant an altered 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 by the introduction of a nucleic acid.

[0126] By the term "modulating" as used herein, it is meant to mediate a detectable increase or decrease in the level of response in a subject, compared to the level of response in a subject in the absence of treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject.This term encompasses disrupting and / or affecting native signals or responses, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0127] In the context of the present invention, the following abbreviations are used for the commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0128] The term "oligonucleotide" typically refers to a short polynucleotide. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it is understood that this also includes an RNA sequence in which "U" is substituted for "T" (i.e., A, U, C, G).

[0129] 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 encoding a protein or RNA can also include introns to the extent that a nucleotide sequence encoding a protein, depending on the type, may contain introns.

[0130] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0131] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, "nucleic acid" and "polynucleotide" as used herein are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides" and contains one or more "nucleotide sequences". The monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotide as used herein includes, but is not limited to, all nucleic acid sequences (i.e., "nucleotide sequences") obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from a cell genome using recombinant libraries or conventional cloning techniques and PCR, etc., and synthetic means.

[0132] The terms "peptide", "polypeptide" and "protein" as used herein are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limit is placed on the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. The term as used herein refers to both short chains, also broadly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, generally 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. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0133] By the term "specifically bind" as used herein with respect to an antibody, it is meant an antibody that recognizes a particular 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. However, such cross-species reactivity does not, in itself, change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not, in itself, change the specific classification of the antibody. In some cases, the term "specifically bind" or "specifically binding" can be used in relation to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than proteins in general. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free unlabeled A) in a reaction containing labeled "A" and an antibody reduces the amount of labeled A bound to the antibody.

[0134] By the term "stimulation" is meant a primary response induced by binding of a stimulatory molecule (e.g., the TCR / CD3 complex) with its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex. Stimulation can mediate changes in expression of certain molecules, such as downregulation of TGF-beta, and / or rearrangements of cytoskeletal structure, among others.

[0135] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0136] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies, among others.

[0137] The term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be elicited. A "subject" or "patient" as used herein can be a human or a non-human mammal. Non-human mammals include, for example, farm animals and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as monkeys and non-human primate mammals. Preferably, the subject is a human.

[0138] "Target site" or "target sequence" refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0139] The term "T cell receptor" or "TCR" as used herein refers to a membrane protein complex involved in the activation of T cells in response to antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells the TCR consists of gamma and delta (γ / δ) 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 domain and a constant domain. In some embodiments, TCRs can be engineered on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0140] The term "therapeutic" as used herein means treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of a disease condition.

[0141] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0142] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0143] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid inside a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and others. Examples of viral vectors include but are not limited to Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and others.

[0144] Ranges: Throughout this disclosure, various aspects of the invention may 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. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0145] B. Chimeric Antigen Receptor The present invention provides modified immune cells or precursor cells thereof (e.g., modified T cells) engineered to express (a) a first chimeric antigen receptor (CAR) comprising (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second CAR comprising (i) a second extracellular antigen binding domain having affinity for a tumor antigen other than CD19, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain. Nucleic acids comprising nucleotide sequences encoding the first and second CARs, vectors comprising the nucleic acids, and modified cells (e.g., modified T cells) comprising the first and second CARs, vectors, and / or nucleic acids are also provided. The antigen binding domain of the first or second CAR is functionally linked to another domain of the CAR for expression in the cell, e.g., a hinge, a transmembrane domain, or an intracellular domain, each of which is described elsewhere herein. In one embodiment, a first nucleotide sequence encoding an antigen-binding domain is operably linked to a second nucleotide sequence encoding a hinge and / or transmembrane domain, and is further operably linked to a third nucleotide sequence encoding an intracellular domain.

[0146] The antigen-binding domain described herein can be combined with any of the transmembrane domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that may be included in the CAR of the present invention, such as a hinge domain or a spacer sequence.The CAR of the present invention may also include a leader sequence.The CAR of the present invention may also include one or more spacer domains or linkers described herein, which may serve to link one domain of the CAR with the next domain.

[0147] Antigen-binding domain The extracellular antigen binding domain of the CAR serves to recognize (i.e., bind to) a specific target antigen, which may include proteins, carbohydrates, and glycolipids. The first CAR of the present invention comprises an extracellular antigen binding domain having affinity for CD19. The first CAR further comprises an intracellular domain comprising a transmembrane domain and at least one co-stimulatory domain and a signaling domain, as described elsewhere herein. In some embodiments, the first CAR comprises a CD28 co-stimulatory domain and a CD3 zeta signaling domain.

[0148] CD19 is naturally expressed on B cells, and because (i) adoptively transferred cells encounter CD19 immediately upon entering the patient's circulation, (ii) there is a large amount of natural CD19 in the body (Morbach, et al., Clinical and Experimental Immunology, 2010), and (iii) CD19 can self-renew from hematopoietic stem cells (HSCs) that differentiate into B cells, it was selected as the antigen to be recognized by the first CAR to "boost" the expansion of CAR T cells in peripheral blood. This can potentially maintain the continued presence of CAR-T cells in the patient as long as the patient has healthy HSCs. Additionally, even if a patient experiences B cell aplasia after receiving anti-CD19 (e.g., CTL019) CAR-T cells, treatment with supplemental immunoglobulin infusions can restore the patient's immunoglobulin levels to normal. Furthermore, large-scale real-world analysis has not revealed a significant risk of infection in patients treated with CTL019 (Schultz, et al., J. Clin. Oncol., 2021:JCO. 20.03585). Anti-CD19 antigen-binding domains are known in the art. For example, anti-CD19 "FMC63" scFv. The first CAR further comprises a transmembrane domain and an intracellular domain comprising at least one co-stimulatory domain and a signaling domain. In some embodiments, the first CAR comprises a CD28 co-stimulatory domain and a CD3 zeta signaling domain.

[0149] The second CAR of the present invention comprises an extracellular antigen binding domain having affinity for a tumor antigen, where the tumor antigen is not CD19. The second CAR further comprises a transmembrane domain and an intracellular domain comprising at least one co-stimulatory domain and a signaling domain as described elsewhere herein. In some embodiments, the second CAR comprises a 4-1BB co-stimulatory domain. In some embodiments, the second CAR comprises a CD3 zeta signaling domain. In some embodiments, the second CAR comprises an intracellular domain comprising a 4-1BB co-stimulatory domain and a CD3 zeta signaling domain. The second CAR of the present invention targets any tumor antigen other than CD19, examples of which include, but are not limited to, CARs that target TnMuc1 (see, e.g., WO2020198413A1), GFRα4 (see, e.g., WO2016025880A1), PSMA (see, e.g., WO2020181094A1), EGFR (see, e.g., WO2021041725A1 and WO2020210768A1), IL13Rα2 (see, e.g., WO2021041725A1 and WO2020210768A1), and mesothelin (see, e.g., WO2017 / 112741).

[0150] Suitable tumor antigens that the second CAR targets (i.e., binds) are known in the art and include alphafetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, 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, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), glycosyl-phosphatidylinositol (GPI)-linked GDNF family receptor 4 (GFR a-4), and α4; GFR alpha 4), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Rα), interleukin 13 receptor subunit alpha 1 (IL13Rα1), interleukin 13 receptor subunit alpha 2 (IL13Rα2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, These include, but are not limited to, MUC1, Nectin4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1-6), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), VEGFR2, and any combination thereof.In some embodiments, the tumor antigen is selected from prostate-specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFRalpha4), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2).

[0151] In one embodiment, the engineered immune cell or progenitor thereof (e.g., engineered T cell) comprises: (a) a first chimeric antigen receptor (CAR) comprising: (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) (i) a second extracellular antigen binding domain having affinity for at least one tumor antigen, (ii) a second transmembrane domain, and (iii) at least one costimulatory domain and a signaling domain. and a second intracellular domain comprising a null transduction domain; wherein the at least one tumor antigen is alpha feto-protein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, 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, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFRalpha4), HER2, HLA-A2, ICAM1, interleukin-13 receptor subunit alpha (IL3Rα), interleukin-13 receptor subunit alpha 1 (IL13Rα1), interleukin-13 receptor subunit alpha 2 (IL13Rα2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin 4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1-6), New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1), P16, PD-L1, Prostate Stem Cell Antigen (PSCA), Prostate Specific Membrane Antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), VEGFR2, and any combination thereof. In some embodiments, the tumor antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFRalpha4), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2).

[0152] In one embodiment, the engineered immune cell or progenitor thereof (e.g., engineered T cell) is engineered to express: (a) a first chimeric antigen receptor (CAR) comprising: (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second CAR comprising: (i) a second extracellular antigen binding domain having affinity for a tumor antigen, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; wherein the tumor antigen is a precursor of the engineered immune cell or progenitor thereof. and selected from the group consisting of prostate-specific membrane antigen (PSMA), MUC1, the Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFRalpha4), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2).

[0153] The antigen-binding domain of the CAR can comprise any domain that binds to an antigen, including, but 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 a scFv thereof.

[0154] The term "single-chain variable fragment" or "scFv" as used herein refers to a fusion protein in which the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an immunoglobulin (e.g., mouse or human) are covalently linked to form a VH::VL heterodimer. The variable heavy chain (VH) and the variable light chain (VL) are directly linked or linked by a peptide linker connecting the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen binding domain (e.g., a tumor antigen binding domain) comprises an scFv having, from the N-terminus to the C-terminus, the VH-linker-VL configuration. In some embodiments, the antigen binding domain comprises an scFv having, from the N-terminus to the C-terminus, the VL-linker-VH or VH-linker-VL configuration. Those skilled in the art can select the appropriate configuration for use in the present invention.

[0155] The linker is usually rich in glycine for flexibility, but also rich in 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 WO2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in the art, including but not limited to glycine serine (GS) linkers. Those skilled in the art can select a suitable linker sequence for use in the present invention. In one embodiment, the 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 the VL are separated by a linker sequence.

[0156] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids comprising VH and VL coding 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 Application Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (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 et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 183(4):2277-85; 2(10:31-40). Agonistic scFvs with 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).

[0157] "Fab" as used herein refers to the fragment of an antibody structure that binds an antigen but is monovalent and does not have the Fc portion; for example, digestion of an antibody with the enzyme papain results in two Fab fragments and an Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind antigen).

[0158] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, where the fragment has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains for binding to the antigen, i.e., a portion of a heavy chain and a light (L) chain linked by an S-S bond, with the remaining heavy chain portions linked together. The "F(ab')2" fragment can be split into two separate Fab' fragments.

[0159] In other embodiments, the antigen binding domain comprises an antibody mimetic protein, such as a designed ankyrin repeat protein (DARPin), an affibody, a monobody, (i.e., an adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, or anticalin. For example, DARPin libraries can be used to generate constructs with specific binding affinities, as described in Seeger, et al., , Protein Sci., 22:1239-1257 (2013).

[0160] In some embodiments, the antigen binding domain can be derived from the same species that the CAR will ultimately be used.For example, for human use, the antigen binding domain of the CAR can comprise a human antibody or a fragment thereof.In some embodiments, the antigen binding domain can be derived from a different species than the species that the CAR will ultimately be used.For example, for human use, the antigen binding domain of the CAR can comprise a mouse antibody or a fragment thereof, or a humanized mouse antibody or a fragment thereof.

[0161] In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs). In some embodiments, the antigen binding domain comprises a linker.

[0162] Transmembrane domain The CAR of the present invention comprises a transmembrane domain that connects the antigen-binding domain of the CAR with the intracellular domain of the CAR. The transmembrane domain of the CAR is a region that can penetrate the plasma membrane of a cell (e.g., an immune cell or its precursor cell). In some embodiments, the transmembrane domain is inserted between the antigen-binding domain and the intracellular domain of the CAR.

[0163] In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, such a domain can be selected or modified by one or more amino acid substitutions to avoid binding of the transmembrane domain with the transmembrane domain of the same or different surface membrane protein and minimize interaction with other members of the receptor complex.

[0164] The transmembrane domain can be derived from either natural or synthetic origin.If the origin is natural, the domain can be derived from any membrane-bound or transmembrane protein, for example, type I transmembrane protein.If the origin is synthetic, the transmembrane domain can be any artificial sequence, for example, an artificial hydrophobic sequence, that facilitates the insertion of CAR into cell membrane. Examples of transmembrane domains that are particularly useful in the present invention include, but are not limited to, a transmembrane domain (i.e., comprising at least the transmembrane region thereof) derived from the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, 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).

[0165] In certain embodiments, the transmembrane domain comprises the transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is the transmembrane domain of CD8α.

[0166] In some embodiments, the transmembrane domain may be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.

[0167] 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, such as a hinge domain or region, that can be included in a CAR.

[0168] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also comprise a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain promotes proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may comprise a domain selected from an antibody Fc fragment, an antibody hinge region, an antibody CH2 region, an antibody CH3 region, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CH1 and CH3 domains of IgG (such as human IgG4), as well as CD8a hinges, artificial hinges made of polypeptides that may be as small as three glycines (Gly).

[0169] In some embodiments, the CAR of the present disclosure comprises a hinge region that connects the antigen binding domain with the transmembrane domain, which in turn connects with the intracellular domain. The hinge region preferably assists the antigen binding domain to recognize and bind to the target antigen on the target cell (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 for optimally recognizing the specific structure and density of the target antigen on a cell, such as a tumor cell (Hudecek et al., supra). The flexibility of the hinge region allows it to adopt many different conformations.

[0170] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0171] The hinge region can have a length of about 4 amino acids to about 50 amino acids, e.g., about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of more than 5 aa, more than 10 aa, more than 15 aa, more than 20 aa, more than 25 aa, more than 30 aa, more than 35 aa, more than 40 aa, more than 45 aa, more than 50 aa, more than 55 aa, or more.

[0172] Suitable hinge regions can be readily selected and can be any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, including from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can be greater than 20 amino acids in length (e.g., 30, 40, 50, 60 or more amino acids).

[0173] For example, the hinge region may be a glycine polymer (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 amorphous and therefore can serve as neutral tethers between components. Glycine polymers can be used; glycine has access to significantly more φ-ψ space than alanine and is much less constrained than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). The hinge region can include the amino acid sequence of the hinge region of human IgG1, IgG2, IgG3, or IgG4 (see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897). In one embodiment, the hinge region may comprise an amino acid sequence derived from human CD8, or a variant thereof.

[0174] Intracellular signaling domains The CAR of the present invention also comprises an intracellular signaling domain. The terms "intracellular signaling domain", "signaling domain", "intracellular domain" and "ICD" are used interchangeably herein. The intracellular signaling domain of the CAR is responsible for the activation of at least one of the effector functions of the cell (e.g., immune cell) in which the CAR is expressed. The intracellular signaling domain transmits the signal of the effector function and instructs the cell (e.g., immune cell) to perform its specialized function (e.g., harm and / or destroy target cells).

[0175] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecule that acts in concert to initiate signaling in an immune cell (i.e., a T cell), as well as any derivatives or variants of these elements, and any synthetic sequences that have the same function.

[0176] Examples of intracellular signaling domains include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologues, e.g., the η chain, FcsRI γ and β chains, MB1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell signaling, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may comprise the intracellular signaling domain (i.e., "ICD") of a protein selected from human CD3 zeta chain, FcyRIII, FcsRI, DAP10, DAP12, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), and combinations thereof.

[0177] In one embodiment, the intracellular signaling domain of the CAR comprises any portion of one or more costimulatory molecules, e.g., at least one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof having the same function, e.g., any synthetic sequence thereof, and any combination thereof.

[0178] Other examples of intracellular domains include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon RIb), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT The co-stimulatory molecules include fragments or domains derived from one or more molecules or receptors, including, but not limited to, AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), 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 having the same function, and any combination thereof.

[0179] Further examples of intracellular domains include, but are not limited to, the 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 can include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), such as the signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0180] Intracellular signaling domains suitable for use in the CARs of the present invention include any desired signaling domain that provides a distinct detectable signal (e.g., increased production of one or more cytokines by the cell; transcriptional changes in target genes; changes in protein activity; changes in cellular behavior, e.g., cell death, cell proliferation, cell differentiation, cell survival; modulation of cellular signaling responses, etc.) in response to activation of the CAR (i.e., activated by antigen and dimerization agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) of the following ITAM motifs. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is not covalently linked to the membrane-bound CAR, but instead is diffused in the cytoplasm.

[0181] Intracellular signaling domains suitable for use in the CARs of the invention comprise an immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptide. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, with the first and second instances of the ITAM motif separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the CAR comprises three ITAM motifs.

[0182] In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0183] Suitable intracellular signaling domains can be ITAM motif-containing moieties derived from ITAM motif-containing polypeptides. For example, suitable intracellular signaling domains can be ITAM motif-containing domains from any ITAM motif-containing protein. Thus, suitable intracellular signaling domains do not need to contain the entire sequence of the entire protein from which they are 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).

[0184] In one aspect, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor associated protein; killer-activating receptor associated protein, etc.). In one aspect, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor high affinity gamma chain, etc.). In one aspect, the intracellular signaling domain is derived from the 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 surface glycoprotein CD3 delta chain, etc.). In one aspect, the intracellular signaling domain is derived from the 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, CD3 epsilon, T3e, etc.). In one aspect, the intracellular signaling domain is derived from the 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 aspect, the intracellular signaling domain is derived from the 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 aspect, 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, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the 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 comprises the cytoplasmic signaling domain of human CD3 zeta.

[0185] Typically, the entire intracellular signaling domain can be used, although 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 a truncated portion can be used in place of the intact chain, so long as it transmits an effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain that is sufficient to transmit an effector function signal.

[0186] 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 can be included in a CAR.

[0187] In some embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB. In some embodiments, the intracellular domain comprises an intracellular domain of CD3zeta or a variant thereof. In some embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB and an intracellular domain of CD3zeta.

[0188] Acceptable variations in individual CAR domain sequences (leader, antigen binding domain, hinge, transmembrane, and / or intracellular domains) are known to those of skill in the art. For example, in certain embodiments, the CAR 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%, or at least 99% sequence identity to any naturally occurring or known sequence.

[0189] Exemplary amino acid and nucleotide sequences for the first and second CARs disclosed herein are as follows: TIFF2025506515000001.tif229150TIFF2025506515000002.tif225150TIFF2025506515 000003.tif223150TIFF2025506515000004.tif223150TIFF2025506515000005.tif86150

[0190] The anti-MSLN scFv is an example of a second extracellular antigen binding domain having affinity for a tumor antigen, where the tumor antigen is not CD19. In some embodiments, the tumor antigen that is not CD19 is mesothelin. In some embodiments, the anti-MSLN scFv comprises an amino acid sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of the M5 human anti-MSLN-scFv. In some embodiments, the anti-MSLN scFv is an M11 human anti-MSLN scFv.

[0191] Further exemplary amino acid and nucleotide sequences for second extracellular antigen-binding domains with affinity for tumor antigens include, but are not limited to, the following: TIFF2025506515000006.tif55149TIFF2025506515000007.tif220149TIFF20255065150 00008.tif216149TIFF2025506515000009.tif215150TIFF2025506515000010.tif29149

[0192] Further CAR domain sequences include: TIFF2025506515000011.tif182150TIFF2025506515000012.tif219150TIFF2025506515000013.tif223150 TIFF2025506515000014.tif211150TIFF2025506515000015.tif226150TIFF2025506515000016.tif159150

[0193] C. Nucleic acids and expression vectors The present disclosure provides an isolated nucleic acid comprising a first nucleotide sequence encoding a first CAR and a second nucleotide sequence encoding a second CAR as described herein. That is, the present invention provides an isolated nucleic acid comprising: (a) a first nucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second nucleotide sequence encoding a second CAR comprising (i) a second extracellular antigen binding domain having affinity for a tumor antigen that is not CD19, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain.

[0194] In one embodiment, the isolated nucleic acid comprises: (a) a first nucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second nucleotide sequence encoding a second CAR comprising (i) a second extracellular antigen binding domain having affinity for a tumor antigen, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and selected from the group consisting of gland-specific membrane antigen (PSMA), MUC1, the Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFR alpha 4), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2).

[0195] In some embodiments, the first nucleotide sequence is located 5' to the second nucleotide sequence. In some embodiments, the first nucleotide sequence is located 3' to the second nucleotide sequence. In some embodiments, the isolated nucleic acid further comprises a linker nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence. A linker for use in the present disclosure allows multiple proteins to be encoded by the same nucleic acid (e.g., a multicistronic or bicistronic sequence), which are translated as a polyprotein that dissociates into separate protein components. In some embodiments, the nucleic acid comprises, from 5' to 3', the first nucleotide sequence, the linker, and the second nucleotide sequence. In some embodiments, the nucleic acid comprises, from 5' to 3', the second nucleotide sequence, the linker, and the first nucleotide sequence.

[0196] In some embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into the start codon, e.g., ATG, of a protein coding region, thereby resulting in cap-independent translation of the gene. A variety of internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRESs that can be obtained from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRESs that can be obtained from, for example, cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those skilled in the art can select a suitable IRES for use in the present invention.

[0197] In some embodiments, the linker comprises a ribosome slippage sequence, also known in the art as a sequence encoding a self-cleaving peptide.As used herein, "self-cleaving peptide" or "2A peptide" refers to a sequence that allows multiple proteins to be encoded as a polyprotein, which dissociates into component proteins upon translation.The use of the term "self-cleavage" is not intended to imply a proteolytic cleavage reaction. A variety of ribosomal slippage sequences, i.e., self-cleaving peptides or 2A peptides, are known to those of skill in the art, including, but not limited to, those found in members of the viral family Picornaviridae, such as foot and mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV), and porcine tessiovirus-1 (PTV-1); and cardioviruses, such as tylovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. One of skill in the art will be able to select a ribosomal slippage sequence suitable for use in the present invention.

[0198] In some embodiments, the construct comprises a linker that may further comprise a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease present in the trans-Golgi that processes and then secretes protein precursors. Furin cleaves at the COOH-terminus of its consensus recognition sequence. A variety of furin consensus recognition sequences (or "furin cleavage sites") are known to those skilled in the art. Those skilled in the art can select a suitable furin cleavage site for use in the present invention.

[0199] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, 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 skilled in the art can select a suitable 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 the 2A peptide. In some embodiments, the linker comprises a 2A peptide 5' to the furin cleavage site. A variety of spacer sequences are known in the art, including, but not limited to, glycine serine (GS) spacers (also known as GS linkers). Those skilled in the art can select a suitable spacer sequence for use in the present invention.

[0200] In some aspects, the nucleic acids of the disclosure may be operably linked to transcriptional control elements, such as promoters, enhancers, etc. Suitable promoter and enhancer elements are known to those of skill in the art.

[0201] In one embodiment, the nucleic acid encoding the exogenous CAR is operably linked to a promoter. In one embodiment, the promoter is a phosphoglycerate kinase-1 (PGK) promoter.

[0202] For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression in eukaryotic cells, 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 promoter; promoters present in long terminal repeats from retroviruses; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated and obtained from many organisms, for example, eukaryotes and prokaryotes. For example, modification of reversible promoters from a first organism for use in a second organism, such as a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, is well known in the art.Such reversible promoters, and systems based on such reversible promoters but also including additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (A1cR), etc.), tetracycline-regulated promoters, (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, 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, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0203] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK cell-specific promoter.For example, the CD4 gene promoter can be used; see, for example, Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416.As another example, the CD8 gene promoter can be used.NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al. Blood (2011) 117:1565.

[0204] For expression in yeast cells, suitable promoters are constitutive promoters, such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, etc.; or regulatable promoters, such as the GAL1 promoter, the GAL10 promoter, the ADH2 promoter, the PHOS promoter, the CUP1 promoter, the GALT promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the 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, the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, e.g., the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, etc.; the araBAD promoter; in vivo regulated promoters, such as the ssaG promoter or related promoters (see, e.g., U.S. Patent Application Publication No. 20040131637), the 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), the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and others (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); sigma70 promoters, e.g., the consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); stationary phase promoters, e.g., the dps promoter, the spv promoter, and the like; promoters from pathogenicity island SPI-2 (see, e.g., WO96 / 17951); actA promoters (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoters (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); tet promoters (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res.(1984) 12:7035); and others. Strong promoters suitable 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 the lactose promoter operator (the LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the 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 the tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25).

[0205] 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 operably linked thereto. Other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, EF-1 alpha (aka EF-1α or EF1α) promoter, as well as human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0206] In some embodiments, the locus or construct or transgene containing a suitable promoter is irreversibly switched by induction of an inducible system. Suitable systems for inducing irreversible switches are well known in the art, for example, induction of irreversible switches may utilize Cre-lox mediated recombination (see, for example, Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc. known in the art may be used to generate irreversibly switchable promoters. Methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein are used to generate irreversibly switched promoters and are well known in the art. See, e.g., Grindley et 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.

[0207] In some embodiments, the 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, for example, Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine that is operably linked to a T cell activation-responsive promoter. In some embodiments, the cytokine that is 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.

[0208] The nucleic acids of the present disclosure may be present in an expression vector and / or a cloning vector. Expression vectors may include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and others. Numerous suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example, but should not be construed as limiting in any way: Bacteria: 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, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0209] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of a nucleic acid sequence encoding a heterologous protein. A selectable marker operative in the expression host may be present. Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (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 viruses (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 International Publication No. 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); retroviral vectors (e.g., viral vectors based on murine leukemia virus, spleen necrosis virus, and retrovirus-derived vectors such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0210] Additional expression vectors suitable for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid virus vectors, transposon-mediated vectors, and others.Viral vector techniques are well known in the art and are described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and other virology and molecular biology manuals.Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0211] Generally, suitable vectors contain 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).

[0212] In some embodiments, an expression vector (e.g., lentiviral vector) can be used to introduce a CAR into immune cells or their precursor cells (e.g., T cells). Thus, an expression vector (e.g., lentiviral vector) of the present invention can include a nucleic acid encoding a CAR. In some embodiments, an expression vector (e.g., lentiviral vector) includes additional elements that aid in the functional expression of a CAR encoded therein. In some embodiments, an expression vector including a nucleic acid encoding a CAR further includes a mammalian promoter. In one embodiment, the vector further includes an elongation factor-1-alpha promoter (EF-1α promoter). Use of the EF-1α promoter may increase the efficiency of expression of a downstream transgene (e.g., a nucleic acid sequence encoding a CAR). A physiological promoter (e.g., EF-1α promoter) may be less likely to induce integration-mediated genotoxicity and may negate the ability of a retroviral vector to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those of skill in the art and may be incorporated into the vectors of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises unnecessary cis-acting sequences that can improve titer and gene expression. A non-limiting example of unnecessary cis-acting sequences is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear import. Other unnecessary cis-acting sequences are known to those skilled in the art and may be incorporated into the vector (e.g., lentiviral vector) of the present invention. In some embodiments, the vector further comprises a post-transcriptional regulatory element. The post-transcriptional regulatory element may improve RNA translation, improve expression of the transgene, and stabilize the RNA transcript. One example of a post-transcriptional regulatory element is the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). Thus, in some embodiments, the vector for the present invention further comprises a WPRE sequence.A variety of post-transcriptional regulatory elements are known to those skilled in the art and may be incorporated into the vectors (e.g., lentiviral vectors) of the present invention. The vectors of the present invention may further comprise additional elements such as rev response element (RRE) for RNA transport, packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeats" or "LTRs" refers to the domains of base pairs located at the ends of retroviral DNA, which include the U3, R and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promotion, initiation and polyadenylation of gene transcripts) and viral replication. In one embodiment, the vectors (e.g., lentiviral vectors) of the present invention comprise 3'U3 deleted LTRs. Thus, the vectors (e.g., lentiviral vectors) of the present invention may comprise any combination of elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector of the invention (e.g., a lentiviral vector) can include, in addition to a nucleic acid encoding a CAR, a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3'U3 deleted LTR'.

[0213] The vector of the present invention can be a self-inactivating vector. The term "self-inactivating vector" as used herein refers to a vector in which the 3'LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). The self-inactivating vector may prevent viral transcription beyond the first round of viral replication. As a result, the self-inactivating vector may be able to infect and then integrate into the host genome (e.g., mammalian genome) only once, and cannot be further passaged. Therefore, the self-inactivating vector may greatly reduce the risk of generating replication-competent virus.

[0214] In some embodiments, the nucleic acid of the present invention can be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled 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, for example, Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a CAR of the present disclosure into a host cell can be performed in vitro, ex vivo, or in vivo. For example, RNA comprising a nucleotide sequence encoding a CAR of the present disclosure can be electroporated into a host cell (e.g., NK cell, cytotoxic T lymphocyte, etc.) in vitro or ex vivo.

[0215] To evaluate the expression of a polypeptide or a portion thereof, the expression vector introduced into the cell may also contain either or both of a selection marker gene or a reporter gene to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected via the viral vector. In some embodiments, the selection marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Appropriate regulatory sequences may flank both the selection marker and the reporter gene to allow expression in the host cell. Useful selection markers include, but are not limited to, antibiotic resistance genes.

[0216] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by recipient organisms or tissues and code for polypeptides whose expression is manifested by some easily detectable property, such as enzymatic activity. The expression of reporter genes is evaluated at a suitable time after DNA is introduced into recipient cells. Suitable reporter genes may include, but are not limited to, genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0217] In some embodiments, the nucleic acid of the present disclosure is provided for the production of the CAR described herein, for example, in mammalian cells.In some embodiments, the nucleic acid of the present disclosure provides for the amplification of the nucleic acid and / or vector encoding the CAR.In some embodiments, a cell is provided that comprises the isolated nucleic acid and / or vector, wherein the cell is selected from bacterial cells, fungal cells, yeast cells, insect cells, animal cells, mammalian cells, and human cells.In some embodiments, the cell is a mammalian or human cell, and is an immune cell or a precursor thereof, for example, a T cell.

[0218] D. Engineered immune cells The present invention further provides modified immune cells or precursor cells thereof engineered to express the first and second CARs of the present invention described herein, i.e., the modified cells are immune cells or precursor cells thereof engineered to express (a) a first chimeric antigen receptor (CAR) comprising (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second CAR comprising (i) a second extracellular antigen binding domain having affinity for a tumor antigen that is not CD19, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain.

[0219] In one aspect, the modified cell is an immune cell, or a precursor thereof, that has been engineered to express: (a) a first chimeric antigen receptor (CAR) comprising: (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second CAR comprising: (i) a second extracellular antigen binding domain having affinity for a tumor antigen that is not CD19, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain.

[0220] In some embodiments, the modified immune cells or precursors thereof are selected from T cells (e.g., including but not limited to, natural killer T (NKT) cells and gamma-delta T cells), natural killer (NK) cells, and macrophages). In some embodiments, the modified cells are autologous cells. In some embodiments, the modified cells are autologous cells obtained from a human subject. In some embodiments, the modified cells are T cells.

[0221] In some embodiments, the modified cells of the present invention are further engineered to express one or more additional receptors. In some embodiments, the modified cells are further engineered to express a switch receptor. In some embodiments, the modified cells are further engineered to express a dominant negative receptor. In some embodiments, the modified cells are further engineered to express a switch receptor and a dominant negative receptor. As used herein, a "switch receptor" is a chimeric molecule that can switch a negative signal to a positive signal for enhanced immune response. See, for example, WO2013019615A2 and WO2016122738A1. A switch receptor is a chimeric protein that includes an extracellular domain of a first protein or a fragment thereof associated with a negative signal, and an intracellular domain of a second protein or a fragment thereof associated with a positive signal. When expressed in a cell, the switch receptor converts a negative signal generated by binding of an immunosuppressive ligand into a positive signal generated by an intracellular signaling domain of a costimulatory molecule. Examples of proteins associated with negative signals include, but are not limited to, CTLA-4, TGFβRII, PD-1, VSIG8, VSIG3, BTLA, and TIM-3. Examples of proteins associated with positive signals include, but are not limited to, CD28, 4-1BB, IL12Rβ1, IL12Rβ2, IL2, IL19, CD2, ICOS, CD27, and others.

[0222] The switch receptor comprises an extracellular domain of a first receptor and an intracellular domain of a second receptor linked by a transmembrane domain. The transmembrane domain of the switch receptor is typically derived from the first receptor or the second receptor, but can be any suitable transmembrane domain. In some embodiments, the first receptor and the second receptor are TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, TIM3 and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, VISTA and IL-9R, TIGIT and IL-9R, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R. In some embodiments, the switch receptor is a TGFβR-IL12R receptor, a TGFβR-CD28 receptor, a TGFβR-OX40 receptor, a PD1-IL12R receptor, a PD1-CD28 receptor, a PD1-ICOS receptor, a PD1-CD27 receptor, a BTLA-CD28 receptor, a BTLA-ICOS receptor, a BTLA-CD27 receptor, an IFNγ-CD28 receptor, an IFNγ-OX40 receptor, or an IFNγ-IL12R receptor.

[0223] In some embodiments, the switch receptor is selected from the group consisting of TGFβR / IL12R, TGFβR / CD28, TGFβR / OX40, PD1 / IL12R, PD1 / CD28, PD1 / ICOS, PD1 / CD27, BTLA / CD28, BTLA / ICOS and BTLA / CD27, IFNγ / CD28, IFNγ / OX40, and IFNγ / IL12R. In some embodiments, the switch receptor is selected from the group consisting of PD-1-CD28, PD-1 A132L -CD28, PD-1 -CD27, PD-1 A132L -CD27, PD-1-4-1BB, PD-1 A132L -4-1BB, PD-1-ICOS, PD-1 A132L -ICOS, PD-1-IL12Rβ1, PD-1 A132L -IL12Rβ1, PD-1-IL12Rβ2, PD-1 A132L-IL12Rβ2, VSIG3-CD28, VSIG8-CD28, VSIG3-CD27, VSIG8-CD27, VSIG3-4-1BB, VSIG8-4-1BB, VSIG3-ICOS, VSIG8-ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG3-IL12Rβ2, VSIG8-IL12Rβ2, VISTA-IL-9R, TIGIT-IL-9R, TGFβRII-CD27, TGFβRII-CD28, TGFβRII-4-1BB, TGFβRII-ICOS, TGFβRII-IL12Rβ1, and TGFβRII-IL12Rβ2. In some embodiments, the switch receptor comprises an extracellular domain of a signaling protein associated with a negative signal, a transmembrane domain, and an intracellular domain of a signaling protein associated with a positive signal. In some embodiments, the transmembrane domain of the switch receptor is selected from a transmembrane domain of a protein associated with negative signaling or a transmembrane domain of a protein associated with negative signaling. In some embodiments, the transmembrane domain of the switch receptor is selected from a transmembrane domain of a protein selected from the group consisting of CTLA4, PD-1, TGFβRII, VSIG8, VSIG3, BTLA, VISTA, TIGIT, IL-9R, IL-2R, TIM-3, CD28, 4-1BB, IL12Rβ1, IL12Rβ2, CD2, ICOS, and CD27.

[0224] Dominant negative (DN) mutants represent an important class of mutations in which DN mutant receptors interfere with the function of wild-type (WT) receptors. Dominant negative receptors as used herein are (a) truncated variants of wild-type receptors, (b) variants of wild-type proteins that include extracellular domains, transmembrane domains, and substantially lack intracellular signaling domains; or (c) the extracellular domains and transmembrane domains of signaling proteins associated with negative signals. In some embodiments, the dominant negative receptors are variants of proteins associated with negative signals. In some embodiments, the dominant negative receptors are selected from CTLA-4, TGFβRII, PD-1, VSIG8, VSIG3, BTLA, and TIM-3 dominant negative receptors. In some embodiments, the dominant negative receptors are dominant negative TGF-β receptors (dnTGFβR). See, for example, WO2016122738A1.

[0225] In some embodiments, the modified cell comprises a vector encoding the first CAR and the second CAR. In some embodiments, the modified cell comprises a vector encoding the first CAR, the second CAR, a switch receptor, and a dominant negative receptor. In some embodiments, the vector is a retroviral vector or a lentiviral vector.

[0226] The modified cells of the present invention can be administered to a subject as a population of modified cells. When administered to a subject, such as a mammal or human, the modified cells show expansion in the peripheral blood of the subject. In some embodiments, the expansion is at least 10-fold, at least 100-fold, or at least 1000-fold. In some embodiments, the modified cells are detectable for at least 24 months after the cells are administered. In some embodiments, the population of modified cells includes T cells, and at least 30% or at least 40% of the population of modified cells after 7 days after administration are phenotypically central memory T cells.

[0227] E. Treatment The present invention involves the use of CD19 antigen-driven expansion of CAR-expressing immune cells (e.g., CAR T cells) in the peripheral blood (PB) of a patient to safely increase the therapeutic index of adoptive cell therapy targeting solid tumors. This is accomplished using a dual CAR approach, in which a subject receives immune cells (i.e., multiple immune cells, e.g., multiple T cells) engineered to express a first CAR that targets the CD19 antigen and a second CAR that targets a tumor antigen other than CD19 (i.e., dual CAR T cells; dual CAR immune cells). The CD19 antigen in the subject drives the expansion of the dual CAR T cells (or the expansion of the CAR immune cells). The CD19 antigen is present endogenously in the subject, e.g., on CD19-expressing B cells, and / or is provided exogenously, e.g., as a CD19 antigen protein, as a CD19 antigen-expressing cell, or as a nucleic acid comprising a nucleotide sequence encoding the CD19 antigen. Compared to cells expressing a single CAR targeting a tumor antigen, the dual CAR T cell approach (dual CAR immune cells) of the present invention surprisingly enhances the in vivo expansion of CAR T cells in both peripheral blood and organs of the subject, enhances and promotes tumor cell killing (e.g., solid tumor cell killing and tumor size reduction) in vitro and in vivo, increases the expression of cytotoxic cytokines, and reduces cytotoxicity against non-tumor normal cells. Importantly, boosting anti-tumor efficacy does not require lymphodepletion. Additionally, the boosted dual CAR T cells of the present invention are primarily effector memory or central memory phenotypes and express only one or two exhaustion markers. Importantly, these unexpected effects are not simply additive effects of combining two CARs, and the present invention addresses a long-standing need to treat solid tumors with adoptive cell therapy.

[0228] The modified cells (e.g., T cells) described herein may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and may further include a pharma- ceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified T cells may be administered.

[0229] In one aspect, the present invention includes a method for adoptive cell transfer therapy, comprising administering to a subject in need thereof a population of modified cells of the present invention, wherein the cells are immune cells or precursor cells thereof (e.g., T cells). In one aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a population of modified cells, wherein the cells are immune cells or precursor cells thereof, and the cells are engineered to express (a) a first chimeric antigen receptor (CAR) comprising (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second CAR comprising (i) a second extracellular antigen binding domain having affinity for a tumor antigen that is not CD19, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain.

[0230] 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 population of modified cells, wherein the cells are immune cells or precursors thereof and comprise: (a) a first chimeric antigen receptor (CAR) comprising (i) a first extracellular antigen binding domain having affinity for CD19, (ii) a first transmembrane domain, and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and (b) a second chimeric antigen receptor (CAR) comprising (i) a second extracellular antigen binding domain having affinity for a tumor antigen, (ii) a second transmembrane domain, and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain. and the tumor antigen is selected from the group consisting of prostate specific membrane antigen (PSMA), MUC1, the Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFRalpha4), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin 13 receptor subunit alpha 1 (IL13Rα1), and interleukin 13 receptor subunit alpha 2 (IL13Rα2).

[0231] In some aspects, the modified cells are further engineered to express a switch receptor described herein, a dominant negative receptor described herein, or both.

[0232] In some embodiments, the modified cell comprises a vector encoding the first CAR and the second CAR. In some embodiments, the modified cell comprises a vector encoding the first CAR, the second CAR, a switch receptor, and a dominant negative receptor. In some embodiments, the vector is a retroviral vector or a lentiviral vector.

[0233] In some embodiments, the population of modified cells is about 1×10 6 ~Approx. 1×10 9 The modified cells include T cells. Upon administration to a subject, e.g., a mammal or human, the modified cells exhibit expansion in the peripheral blood of the subject. In some embodiments, the expansion is at least 10-fold, at least 100-fold, or at least 1000-fold. In some embodiments, the modified cells are detectable for at least 24 months after the cells are administered. In some embodiments, the population of modified cells includes T cells, and at least 30% or at least 40% of the population of modified cells 7 days or more after administration are phenotypically central memory T cells.

[0234] In some embodiments, the method further comprises administering a CD19 antigen to the subject. In some embodiments, administering the CD19 antigen comprises administering a CD19 antigen protein, a cell expressing the CD19 antigen, or a nucleic acid comprising a nucleotide sequence encoding the CD19 antigen. In some embodiments, administering the CD19 antigen comprises administering a vector comprising a nucleotide sequence encoding the CD19 antigen or a cell engineered to express the CD19 antigen. The CD19 antigen comprises the CD19 extracellular domain or an antigenic fragment thereof. The CD19 antigen is administered prior to, simultaneously with, or after administration of the population of modified cells. In some embodiments, the vector comprising a nucleotide sequence encoding the CD19 antigen is an adenoviral vector. Any CD19 antigen (i.e., full-length CD19 or an extracellular antigenic fragment thereof) may be used. In some embodiments, the CD19 antigen may be encoded by the following nucleotide sequence and comprises the following amino acid sequence: TIFF2025506515000017.tif125150

[0235] In some embodiments, the method further comprises administering an anti-PD1 immunotherapy to the subject. In some embodiments, the anti-PD1 immunotherapy is an anti-PD1 antibody, such as a monoclonal anti-PD1 antibody, examples of which include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, and dostarlimab. The anti-PD1 immunotherapy is administered prior to, simultaneously with, or after administration of the population of modified cells.

[0236] Methods for administration of immune cells for adoptive cell therapy are known and may be used in conjunction with the provided methods and compositions.For example, adoptive cell therapy is described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). For example, 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. In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by autologous transfer, where cells are isolated and / or otherwise prepared from the subject to be treated with cell therapy or from a sample derived from such a subject.Thus, in some aspects, the cells are derived from a subject, e.g., a patient, in need of treatment and after isolation and processing the cells are administered to the same subject.

[0237] In some embodiments, cell therapy, such as adoptive T cell therapy, is carried out by allogeneic transfer, in which cells are isolated and / or otherwise prepared from a subject other than the subject that will or will eventually receive cell therapy, such as a first subject.In such embodiments, the cells are then administered to a different subject of the same species, such as a second subject.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.

[0238] In some embodiments, the subject is treated with a therapeutic agent that targets a disease or condition, such as a tumor, before administering the cell or cell-containing composition.In some aspects, the subject is resistant or refractory to the other therapeutic agent.In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, including, for example, chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT.In some embodiments, the administration effectively treats the subject even though the subject has become resistant to another therapy.

[0239] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces the disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits recurrence of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject has been determined to be at risk of relapse, such as at high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of relapse or to prevent relapse. In some aspects, the subject has not been previously treated with another therapeutic agent.

[0240] In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, including chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become refractory to another therapy.

[0241] The modified immune cells of the present invention can be administered to animals, preferably mammals, and even more preferably humans, to treat cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to cancer, particularly for cellular immune response against tumor cells, when it is desired to treat or alleviate the disease. The types of cancer to be treated with the modified cells or pharmaceutical compositions of the present invention include certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Exemplary cancers include, but are not limited to, colon cancer, breast cancer, ovarian cancer, renal cancer, non-small cell lung cancer, melanoma, lymphoma, and hepatocellular carcinoma, as well as B-cell malignancies such as B-cell lymphomas and leukemias, etc. The cancer can be a non-solid tumor (such as a hematological tumor) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor. In one embodiment, the cancer is a leukemia and / or lymphoma. In certain embodiments, the cancer cells express CD19.

[0242] The cells to be administered may be autologous with respect to the subject being treated.

[0243] Administration of the cells of the present invention may be carried out in any convenient manner known to those skilled in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In other examples, the cells of the present invention are directly injected into a site of inflammation in a subject, a site of local disease in a subject, a lymph node, an organ, a tumor, etc.

[0244] 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 types and / or a desired ratio of cell types. Thus, the dosage of cells is in some embodiments based on the total number of cells (or number per kg of body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, the dosage of cells is based on the desired total number of cells (or number per kg of body weight) in individual populations or individual cell types. In some embodiments, the dosage is based on a combination of these features, such as the desired total number of cells, the desired ratio, and the desired total number of cells in individual populations.

[0245] In some embodiments, CD8 + and CD4 + A population or subtype of cells, such as T cells, is administered at a desired dose of total cells, such as a desired dose of T cells, or within a tolerance thereof. In some aspects, the desired dose is a desired number of cells, or a desired number of cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is a minimum number of cells or a minimum number of cells per unit body weight, or greater. In some aspects, among the total cells administered at the desired dose, an individual population or subtype is administered at a desired output ratio (e.g., CD4 + vs. CD8 + ratio), e.g., within a certain tolerance or error of such ratio.

[0246] In some embodiments, the cells are administered at or within a tolerance of a desired dose of one or more of the individual populations or subtypes of cells, e.g., a desired dose of CD4+ cells and / or a desired dose of CD8+ cells. In some aspects, the desired dose is the desired number of cells of a subtype or population, or the desired number of such cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is or is greater than the minimum number of cells of a population or subtype, or the minimum number of cells of a population or subtype per unit body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose and a desired ratio of total cells, and / or based on a desired fixed dose of one or more, e.g., each, of the individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and CD4 + vs. CD8 + Based on the desired ratio of cells and / or CD4 + and / or CD8 + Based on desired fixation or minimum dose of cells.

[0247] In certain embodiments, a distinct population of cells, or subtypes of cells, may range from about 1 million to about 100 billion cells, such as, for example, 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, for example, 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, or a range defined by any two of the foregoing values). 0 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 between these ranges.

[0248] In some embodiments, the dose of total cells and / or the dose of individual subpopulations of cells is greater than or equal to 1×10 5 Cells / kg or approximately 1 x 10 5 Cells / kg to approximately 1 x 10 11 cells / kg, 10 4 From 10 11 cells / kilogram (kg) body weight or approximately 10 11 cells / kilogram (kg) body weight, e.g. 10 5 ~10 6 In the range of cells / kg body weight, e.g., 1×10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg or 1 x 10 6 cells / kg body weight, or approximately 1 x 10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg or 1 x 10 6 cells / kg body weight. For example, in some embodiments, the cells are 4 or about 10 4 From 109 or about 10 9 T cells / kilogram (kg) body weight, e.g., 10 5 ~10 6 T cells / kg body weight, e.g., 1 x 10 5 cells / kg T cells, 1.5 x 10 5 cells / kg T cells, 2 x 10 5 cells / kg T cells or 1 × 10 6 cells / kg T cells, or approximately 1 × 10 5 cells / kg T cells, 1.5 x 10 5 cells / kg T cells, 2 x 10 5 cells / kg T cells or 1 × 10 6 In another exemplary embodiment, the modified cells suitable for use in the methods of the present disclosure are administered at about 1×10 5 cells / kg ~ approx. 1×10 6 cells / kg, approximately 1×10 6 cells / kg ~ approx. 1×10 7 cells / kg, approximately 1×10 7 cells / kg ~ approx. 1×10 8 cells / kg, approximately 1×10 8 cells / kg ~ approx. 1×10 9 cells / kg, approximately 1×10 9 cells / kg ~ approx. 1×10 10 cells / kg, approximately 1×10 10 cells / kg ~ approx. 1×10 11 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 1×10 cells / kg. 8 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 1×10 7 In other embodiments, the appropriate dosage is about 1×10 7 Total cells ~ approximately 5 x 10 7 In some embodiments, a suitable dosage is about 1×10 8 Total cells ~ approximately 5 x 10 8 In some embodiments, a suitable dosage is about 1.4×10 total cells. 7Total cells ~ approx. 1.1 x 10 9 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 7×10 9 The total number of cells is 1.

[0249] In some embodiments, the cells are 4 or about 10 4 From 10 9 or about 10 9 CD4 cells / kilogram (kg) body weight + and / or CD8 + Cells, e.g., 10 5 ~10 6 CD4 cells / kg body weight + and / or CD8 + Cells, e.g., 1 x 10 5 CD4 cells / kg + and / or CD8 + cells, 1.5 x 10 5 CD4 cells / kg + and / or CD8 + cells, 2 x 10 5 CD4 cells / kg + and / or CD8 + cells or 1×10 6 CD4 cells / kg body weight + and / or CD8 + cells, or approximately 1 x 10 5 CD4 cells / kg + and / or CD8 + cells, 1.5 x 10 5 CD4 cells / kg + and / or CD8 + cells, 2 x 10 5 CD4 cells / kg + and / or CD8 + cells or 1×10 6 CD4 cells / kg body weight + and / or CD8 + In some embodiments, the cells are administered at about 1×10 6 More than 2.5 x 10 pieces 6 More than 5 x 10 pieces6 More than 7.5 x 10 pieces 6 More than 9 x 10 6 More than 100 CD4 + cells, and / or at least about 1 x 10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 CD4 + cells, and / or at least about 1×10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 CD8+ cells and / or at least about 1×10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 Pieces, or about 9 x 10 6 In some embodiments, the cells are administered at about 10 T cells or within a certain margin of error therebetween. 8 ~10 12 Pieces or about 10 10 ~10 11 T cells, approximately 10 8 ~10 12 Pieces or about 10 10 ~10 11 CD4 + cells, and / or about 10 8 ~10 12 Pieces or about 10 10 ~10 11 CD8 + The cells are administered at or within a certain margin of error thereof.

[0250] In some embodiments, cells are administered at a desired output ratio, or within a tolerance range, of multiple cell populations or subtypes, e.g., CD4+ and CD8+ cells or subtypes. 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., CD4+ Cells vs. CD8 + The ratio of cells) may be from 5:1 or about 5:1, 5:1 or about 5:1 (or from about 1:5 to less than about 5:1), or from 1:3 or about 1:3 to 3:1 or about 3:1 (or from about 1:3 to less than about 3:1), such as from 2:1 or about 2:1 to 1:5 or about 1:5 (or from about 1:5 to less than about 2:1, such as 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, 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 1:1.8, 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 tolerance 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 between these ranges.

[0251] In some embodiments, the dose of modified cells is administered to the subject in need thereof once or multiple times.In some embodiments, the dose of modified cells is administered multiple times, for example, 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 exemplary embodiments, a single dose of modified cells is administered to the subject in need thereof.In exemplary embodiments, a single dose of modified cells is administered to the subject in need thereof by rapid intravenous infusion.

[0252] For prevention or treatment of disease, the appropriate dosage will depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, previous treatments, the subject's clinical history and response to the cells, and the judgment 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.

[0253] In some embodiments, the cells are administered as part of a combination treatment, e.g., simultaneously or sequentially in any order with another therapeutic intervention, e.g., an antibody or engineered cell or receptor, or an agent, e.g., a cytotoxic agent or therapeutic agent. In some embodiments, the cells are co-administered simultaneously or sequentially in any order with one or more additional therapeutic agents or in relation to another therapeutic intervention. In some situations, the cells are co-administered with another therapy close enough in time that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered before one or more additional therapeutic agents. In some embodiments, the cells are administered after one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, e.g., IL-2, to enhance persistence. In some embodiments, the method includes administration of a chemotherapeutic agent.

[0254] In some embodiments, the modified cells of the present invention (e.g., modified cells comprising a CAR) can be administered to a subject in combination with an immune checkpoint antibody (e.g., anti-PD1, anti-CTLA-4, or anti-PDL1 antibody). For example, the modified cells can be administered in combination with, for example, an antibody or antibody fragment that targets PD-1 (programmed cell death 1 protein). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly known as lambrolizumab, also known as MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®), or an antigen-binding fragment thereof. In some embodiments, the modified cells can be administered in combination with an anti-PD-L1 antibody, or an antigen-binding fragment thereof. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, atezolizumab), and MEDI4736 (durvalumab, Imfinzi). In some embodiments, the modified cells may be administered in combination with an anti-CTLA-4 antibody or an antigen-binding fragment thereof. Examples of anti-CTLA-4 antibodies include, but are 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. An immune checkpoint modulator may be administered before, after, or simultaneously with the modified cells comprising the CAR. In some embodiments, a combination treatment including an immune checkpoint modulator may increase the therapeutic efficacy of a therapy including the modified cells of the present invention.

[0255] Following administration of the cells, the biological activity of the engineered cell population is measured in some embodiments, e.g., by any of several known methods. Parameters to be evaluated include specific binding of engineered or natural T cells or other immune cells to an antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in 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 the expression and / or secretion of one or more cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, the biological activity is measured by evaluating clinical outcomes, such as reduction in tumor burden or load.

[0256] In some embodiments, the subject is provided with a second treatment, including but not limited to chemotherapy, radiation therapy, surgery, and drug therapy.

[0257] In some embodiments, the subject can be administered a conditioning therapy prior to 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 a preferred embodiment, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. The administration of a conditioning therapy prior to CAR T cell therapy may increase the efficacy of CAR T cell therapy. Methods for conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, the entirety of which is incorporated herein by reference.

[0258] In some embodiments, the specific dosing regimens of the present disclosure include a lymphodepletion step prior to administration of the modified T cells. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0259] In some embodiments, the lymphocyte depletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m 2 In some embodiments, the lymphodepletion step is about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m 2 / day.

[0260] In some embodiments, the lymphocyte depletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day), and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, the lymphodepletion step comprises administration of fludarabine at a dose of about 300 mg / m 2 / day, and cyclophosphamide at a dose of approximately 30 mg / m 2 The treatment includes administration of fludarabine at a dose of 100 mg / day.

[0261] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m 2 / day for 3 days, and the fludarabine dosage was 30 mg / m 2 / day for 3 days.

[0262] Dosing of lymphodepleting chemotherapy may be scheduled for days -6 to -4 (with a -1 day window, i.e., dosing on days -7 to -5) relative to infusion of T cells (e.g., CAR-T, TCR-T, modified T cells, etc.) on day 0.

[0263] In an exemplary embodiment for a subject with cancer, the subject is administered 300 mg / m 200 mg / ml ... 2 In an exemplary embodiment for subjects with cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at 300 mg / m2 via intravenous infusion three days prior to administration of the modified T cells. 2 The patient will undergo lymphodepleting chemotherapy, including cyclophosphamide.

[0264] In an exemplary embodiment for a subject with cancer, the subject receives about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 In an exemplary embodiment for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m 2 Patients will receive lymphodepleting chemotherapy for three days, including chemotherapy at a dose of

[0265] In an exemplary embodiment for a subject with cancer, the subject receives about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day), and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 In an exemplary embodiment for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of about 300 mg / m 2 / day and cyclophosphamide at a dose of 30 mg / m 2 Patients will receive lymphodepleting chemotherapy containing fludarabine at a dose of 10 mg / kg for 3 days.

[0266] The cells of the present invention can be administered at dosages and routes and times to be determined in appropriate preclinical and clinical experiments and trials. The cell composition may be administered multiple times at dosages within these ranges. The administration of the cells of the present invention may be combined with other methods useful for treating the desired disease or condition as determined by those skilled in the art.

[0267] It is known in the art that one of the adverse effects after infusion of CAR T cells is the onset of immune activation known as cytokine release syndrome (CRS). CRS is immune activation resulting in high inflammatory cytokines. CRS is a known on-target toxicity, the occurrence of which may correlate with efficacy. Clinical and laboratory measures range from mild CRS (systemic 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 leakage, cardiac dysfunction, renal impairment, liver failure, and disseminated intravascular coagulation. Dramatic elevation of cytokines including interferon-gamma, granulocyte-macrophage colony-stimulating factor, IL-10, and IL-6 has been shown after infusion of CAR T cells. One CRS signature is elevated cytokines including IL-6 (severely elevated), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations of clinically available inflammatory markers including ferritin and C-reactive protein (CRP) have also been observed to correlate with CRS syndrome. The presence of CRS generally correlates with the expansion proliferation and progressive immune activation of adoptively transferred cells. It has been demonstrated that the severity of CRS depends on the disease burden at the time of infusion, as patients with high tumor burden experience greater sCRS.

[0268] Therefore, the present invention provides a CRS management strategy suitable for alleviating the physiological symptoms of uncontrolled inflammation after the diagnosis of CRS without compromising the anti-tumor efficacy of engineered cells (e.g., CAR T cells).CRS management strategies are known in the art.For example, systemic corticosteroids may be administered to rapidly reverse the symptoms of sCRS (e.g., grade 3 CRS) without compromising initial anti-tumor response.

[0269] In some embodiments, anti-IL-6R antibody may be administered.An example of anti-IL-6R antibody is tocilizumab, also known as atlizumab, which is a monoclonal antibody approved by the Food and Drug Administration (commercially available as Actemra or RoActemra).Tocilizumab is a humanized monoclonal antibody against interleukin-6 receptor (IL-6R).Administration of tocilizumab has demonstrated almost immediate regression of CRS.

[0270] CRS is generally managed based on the severity of symptoms observed and, in such cases, interventions are adjusted. Decisions to manage CRS may be based on clinical signs and symptoms and response to interventions, rather than just laboratory values.

[0271] Mild to moderate cases are generally treated with symptom management with fluid therapy, nonsteroidal anti-inflammatory drugs (NSAIDs) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability; administration of tocilizumab is recommended. First-line management of CRS may be tocilizumab (not to exceed 800 mg / dose) at a labelled dose of 8 mg / kg IV over 60 minutes in some embodiments; tocilizumab may be repeated Q8 hours. If the response to the first dose of tocilizumab is suboptimal, additional doses of tocilizumab may be considered. Tocilizumab may be administered alone or in combination with corticosteroid therapy. Patients with persistent or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours, or poor response to tocilizumab may be treated with high-dose corticosteroid therapy, typically 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 CRS. Management guidance for CRS 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).

[0272] Concurrent with the clinical manifestations of CRS, features consistent with macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007). MAS appears to be a response to immune activation resulting from CRS and should therefore be considered as a manifestation of CRS. MAS is similar to HLH, which is also a response to immune stimulation. The clinical syndrome of MAS is characterized by high-grade unremitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. This is accompanied by high serum ferritin, soluble interleukin-2 receptor, and triglycerides, as well as reduced circulating natural killer (NK) activity.

[0273] 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 modified immune cells or progenitor cells disclosed herein. Yet another aspect of the invention includes a method of treating cancer in a subject in need thereof comprising administering to the subject modified immune cells or progenitor cells generated by any one of the methods disclosed herein.

[0274] F. Source of immune cells In some embodiments, the source of immune cells (e.g., T cells) is obtained from a subject for ex vivo manipulation and / or in vivo transduction. The source of target cells for ex vivo manipulation may also include, for example, autologous or heterologous donor blood, umbilical cord blood, or bone marrow. For example, the source of immune cells may be derived from the subject that will be treated with the modified immune cells of the present invention, such as the subject's blood, the subject's umbilical cord blood, or the subject's bone marrow. Non-limiting 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 expression are described, for example, 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.

[0275] Immune cells can be obtained from several 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 innate or adaptive immunity, for example, lymphocytes, typically myeloid or lymphoid cells, including 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 respect to the subject to be treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, for example, cells directly isolated from the subject and / or cells isolated from the subject and frozen.

[0276] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), 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 cell is a monocyte or granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In embodiments, a target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell that has been generated from a subject and engineered to alter (e.g., induce mutations in) or engineer expression of one or more target genes and differentiated, e.g., into a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0277] In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the entire T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, e.g., those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, 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, mucosal-associated invariant T (MAIT) cells, innate 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 some embodiments, any number of T cell lines available in the art may be used.

[0278] In some embodiments, the method includes isolating immune cells from a subject, preparing, treating, culturing, and / or manipulating them. In some embodiments, the preparation of the engineered cells includes one or more culturing and / or preparation steps. The cells for manipulation as described may be isolated from a sample, e.g., a biological sample, e.g., a sample obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject having a disease or condition or in need of cell therapy or to whom cell therapy is administered. The subject is, in some embodiments, a human in need of a particular therapeutic intervention, e.g., adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated. Thus, the cells are, in some embodiments, primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that has been 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.

[0279] In some aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0280] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. The cells are obtained in some embodiments from heterologous sources, e.g., mouse, rat, non-human primate, and pig. In some embodiments, the isolation of the cells includes one or more preparative and / or non-affinity-based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove undesired components, to enrich for desired components, to lyse or remove cells sensitive to a particular reagent. In some examples, the cells are separated based on one or more properties, e.g., density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0281] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed, for example to remove the plasma fraction, and the cells are placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing. In some embodiments, components of the blood cell sample are removed and the cells are resuspended directly in medium. In some embodiments, the methods include density-based cell separation methods, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0282] In one embodiment, immune cells are obtained from the circulating blood of an individual, obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or medium, such as phosphate-buffered saline (PBS), for subsequent processing steps, or in a washing solution that is calcium-deficient, may be magnesium-deficient, or may be deficient in many, if not all, divalent cations. After washing, cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed and the cells may be resuspended directly in the medium.

[0283] In some embodiments, the isolation method comprises the separation of different cell types based on the expression or presence of one or more specific molecules in cells, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers may be used. In some embodiments, the separation is affinity or immunoaffinity based separation. For example, in some aspects, the isolation comprises the separation of cells and cell populations based on the expression or expression level of one or more markers of cells, typically cell surface markers, for example by incubation with an antibody or binding partner that specifically binds to such markers, typically followed by a washing step, and separation of the cells that bind to the antibody or binding partner from the cells that do not bind to the antibody or binding partner.

[0284] Such separation steps can be based on positive selection, where cells bound to the reagent are retained for further use, and / or negative selection, where cells 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 when antibodies that specifically identify cell types in a heterogeneous population are not available, so that separation is best performed based on markers expressed by cells other than the desired population. Separation does not have to result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment for a particular type of cell, e.g., cells expressing a marker, refers to increasing the number or percentage of such cells, but does not have to result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, e.g., cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not have to result in the complete removal of all such cells.

[0285] In some examples, multiple rounds of separation steps are performed, and the fractions that have undergone positive or negative selection from one step are 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, for example, by incubating cells with multiple antibodies or binding partners, each specific to a marker targeted for negative selection. Similarly, multiple cell types can be subjected to positive selection simultaneously by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0286] In some embodiments, one or more of the T cell populations are positive for (marker+) or express high levels of one or more particular markers, e.g., surface markers (marker+). 高) cells, or cells that are negative for one or more markers (marker-) or have relatively low levels of it (marker 低 ) expressing cells are enriched or depleted. For example, in some aspects, specific subpopulations of T cells, e.g., cells that are positive for or express 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 markers that are absent or expressed at relatively low levels on some T cell populations (e.g., non-memory cells) but present or expressed at relatively high levels on some other T cell populations (e.g., memory cells). In one embodiment, the cells (e.g., CD8+ cells or T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, and / or are depleted for (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, the cells are enriched for or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some examples, the CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L. For example, positive selection of CD3+, CD28+ T cells can be performed using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0287] In some embodiments, T cells are separated from PBMC samples by negative selection of markers expressed on non-T cells, e.g., B cells, monocytes, or other leukocytes, e.g., CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper T cells from CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or relatively highly expressed on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is performed to increase potency, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects is particularly strong for such subpopulations. In some embodiments, combining TCM-enriched CD8+ T cells with CD4+ T cells further enhances efficacy.

[0288] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. For example, anti-CD8 and anti-CD62L antibodies can be used to enrich or deplete CD62L-CD8+ and / or CD62L+CD8+ fractions from PBMCs. In some embodiments, CD4+ T cell populations and CD8+ T cell subpopulations, such as central memory (TCM) cells, are enriched subpopulations. In some embodiments, enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells expressing or high expressing CD45RA and / or granzyme B. In some aspects, the isolation of CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of 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 negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are carried out simultaneously in some aspects, and sequentially in any order in other aspects. In some aspects, the same CD4 expression-based selection step used to prepare a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, so that both the positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more additional positive or negative selection steps.

[0289] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations with 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, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CDllb, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are coupled to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for the separation of cells for positive and / or negative selection.

[0290] In some embodiments, the cells are incubated and / or cultured prior to or in association with genetic engineering. The incubation step can include culturing, cultivating, stimulating, activating, and / or propagating. In some embodiments, the composition or cells are incubated in the presence of stimulatory conditions or stimulants. Such conditions include conditions designed to induce proliferation, expansion, activation, and / or survival of cells in a population, mimic antigen exposure, and / or prime cells for genetic engineering, e.g., introduction of a recombinant antigen receptor. Conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells. In some embodiments, the stimulatory conditions or agents include one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent turns on or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies, such as antibodies specific for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, e.g., those bound to a solid support, e.g., beads, and / or one or more cytokines. Optionally, the expansion method can further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent includes IL-2 and / or IL-15, e.g., an IL-2 concentration of at least about 10 units / mL.

[0291] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation over a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord. In either case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0292] Cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including but not limited to CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using isolated antibodies, antibody-containing biological samples such as ascites fluid, antibodies bound to a physical support, and cell-bound antibodies.

[0293] Enrichment of the T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can be used to enrich the T cell population for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0294] To isolate a desired cell population by positive or negative selection, the concentration and surface of cells (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (i.e., increase cell concentration) to ensure maximum contact between cells and beads, for example, in one embodiment, a cell concentration of 2 billion cells / ml is used. In one embodiment, a cell concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml are used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, cell concentrations of 125 or 150 million cells / ml can be used. Using higher concentrations can result in increased cell yield, cell activation, and cell expansion.

[0295] T cells can also be frozen after a washing step that does not require a monocyte removal step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and useful in this regard, in a non-limiting example, one method involves using PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin. The cells are then frozen to -80°C at a rate of 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank. Immediate uncontrolled freezing at -20°C or in liquid nitrogen may be used, as well as other methods of controlled freezing.

[0296] In one embodiment, the T cell population is comprised within cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations, and T cell lines. In another embodiment, peripheral blood mononuclear cells comprise the T cell population. In yet another embodiment, purified T cells comprise the T cell population.

[0297] In some embodiments, regulatory T cells (Tregs) can be isolated from the sample. The sample can include, but is not limited to, umbilical cord blood or peripheral blood. In some embodiments, Tregs are isolated by flow cytometry sorting. The sample can be enriched for Tregs before isolation by any means known in the art. The isolated Tregs can be cryopreserved and / or expanded before use. Methods for isolating Tregs are described in U.S. Patent Nos. 7,754,482, 8,722,400, and 9,555,105, and U.S. Patent Application No. 13 / 639,927, the contents of which are incorporated herein in their entirety.

[0298] G. Expansion of immune cells Either before or after the cells are modified to express a CAR, see, e.g., 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; 7,124,576; The cells can be activated and expanded in number using methods described in US Patent Publication Nos. 172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and US Patent Publication No. 20060121005. For example, the T cells of the present invention can be expanded by contact with a surface to which is attached an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface. In particular, the T cell population can be stimulated by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of accessory molecules on the T cell surface, a ligand that binds to the accessory molecule is used. For example, T cells can be contacted with anti-CD3 antibody and anti-CD28 antibody under suitable conditions to stimulate T cell proliferation. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can be used in the present invention, as well as other methods and reagents known in the art (see, for example, 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).

[0299] Expanding T cells by the methods disclosed herein can be about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, and any and all whole integers and partial integers therebetween. In one embodiment, T cells are expanded in the range of about 20-fold to about 50-fold.

[0300] Following culturing, the T cells can be incubated in the cell medium in the culture device for a period of time or until the cells reach confluence or a high cell density for optimal passaging before passaging the cells to another culture device. The culture device can be any culture device commonly used for in vitro culturing of cells. Preferably, the level of confluence is 70% or greater before passaging the cells to another culture device. More preferably, the level of confluence is 90% or greater. The period of time can be any time suitable for in vitro cell culture. 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 2-3 days. The T cells are then harvested from the culture device, from which the T cells can be used immediately or cryopreserved and stored for later use. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing the nucleic acid into the T cells.

[0301] 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 RNA encoding the chimeric membrane protein.

[0302] Another procedure for ex vivo expansion of cells is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 can be an alternative or an addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of T cells includes the addition of cell growth factors or other factors such as those described in U.S. Patent No. 5,199,942, such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding T cells includes culturing T cells with factors selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.

[0303] The culturing step described herein (following contact with an agent described herein or electroporation) can be very short, e.g., less than 24 hours, e.g., 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 further described herein (contact with an agent described herein) can be longer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or more.

[0304] Various terms are used to describe cultured cells. Cell culture generally refers to cells obtained from a living organism and grown under controlled conditions. Primary cell cultures are cultures of cells, tissues, or organs obtained directly from an organism before the first subculture. Cells are expanded in culture when placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger cell population. When cells are expanded in culture, the growth rate of the cells is typically measured by the time required for the cells to double in number, otherwise known as the doubling time.

[0305] Each round of subculture is called a passage. When cells are subcultured, they are called passaged. A particular cell population or cell line is sometimes called or characterized by the number of times they have been subcultured. For example, a cultured cell population that has been subcultured 10 times may be called a P10 culture. The primary culture, i.e., the first culture after isolating cells from tissue, is called P0. After the first subculture, the cells are called a secondary culture (P1 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 skilled in the art that there may be many population doublings during the subculture period; therefore, the population doubling number of a culture is greater than the number of passages. The expansion growth (i.e., the number of population doublings) of cells during the period between subcultures depends on many factors, including but not limited to the seeding density, substrate, medium, and subculture interval.

[0306] In one embodiment, cells may be cultured for a few hours (about 3 hours) to about 14 days or any integer value of time therebetween. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-α, or any other additive for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The medium can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a predetermined set of hormones and / or cytokines in sufficient amounts for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures, not in the cultures of cells that will be infused into the subject. Target cells are maintained under the necessary conditions to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2).

[0307] The medium used to culture the T cells may contain an agent capable of costimulating the T cells. For example, an agent capable of stimulating CD3 is an antibody against CD3, and an agent capable of stimulating CD28 is an antibody against CD28. Cells isolated by the methods disclosed herein can be expanded about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more. In one embodiment, T cells are expanded in the range of about 20-fold to about 50-fold or more. In one embodiment, human regulatory T 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 Nos. 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entirety.

[0308] In one embodiment, the method of expanding T cells can further include isolating the expanded T cells for further application. In another embodiment, the method of expanding can further include subsequent electroporation of the expanded T cells followed by culturing. Subsequent electroporation can include introducing a nucleic acid encoding an agent, for example, transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid to form an expanded T cell population, where the agent further stimulates the T cells. The agent can stimulate the T cells, for example, by stimulating further expansion, effector function, or another T cell function.

[0309] H. Pharmaceutical Compositions and Formulations Also provided are immune cell populations of the present invention, compositions containing such cells and / or enriched with such cells, for example, compositions in which CAR-expressing cells account for at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells or a certain type of cells, such as T cells or CD8+ cells or CD4+ cells, in the composition. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering cells and compositions to subjects, for example, patients.

[0310] Also provided are compositions that contain cells for administration, including pharmaceutical compositions and formulations, for example, unit dose form compositions that contain the number of cells for administration at a given dose or a fraction thereof.Pharmaceutical compositions and formulations generally contain one or more optional pharma-ceutically acceptable carriers or excipients.In some embodiments, the composition contains at least one additional therapeutic agent.

[0311] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in such a form that allows the biological activity of the active ingredient contained therein to be effective, and does not contain additional components that are unacceptably toxic to the subject to which the formulation will be administered. A "pharmaceutical acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some aspects, the choice of carrier is determined, in part, by the particular cell and / or by the method of administration. Thus, there is a wide variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can 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 typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in 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 octadecyldimethylbenzylammonium 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; 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 counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0312] In some aspects, a buffering agent is included in the composition. 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, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0313] The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for a particular indication, disease, or condition treated by the cells, preferably active ingredients with complementary activities to the cells, each of which does not adversely affect the other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmacologic active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition, in some embodiments, contains the cells in an amount effective to treat or prevent a disease or condition, such as a therapeutically or prophylactically effective amount. The therapeutic or prophylactic effectiveness is, in some embodiments, monitored by periodically evaluating the subject being treated. The desired dosage can be delivered by administration of a single bolus of cells, by administration of multiple boluses of cells, or by administration of continuous infusions of cells.

[0314] Formulations include formulations for oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is 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 a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. The composition is provided in some embodiments as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0315] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, for example, in a solvent mixed with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents and / or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard textbooks may be consulted for preparing appropriate preparations.

[0316] Various additives can be added to enhance the stability and sterility of the composition, including antibacterial preservatives, antioxidants, chelating agents, and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0317] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0318] The contents of the articles, patents and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein 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.

[0319] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications and equivalent substitutions may be made without departing from the true spirit and scope of the present 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 appropriate 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. Although certain embodiments have been described in detail herein, the same will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0320] Experimental Examples The invention will now be described with reference to the following examples, which are provided for purposes of illustration only, and the invention is not limited to these examples, but rather includes all variations that are evident as a result of the teachings provided herein.

[0321] material and method mouse Mice were housed in an AAALAC-accredited animal facility under specific pathogen-free conditions at the University of Pennsylvania (Upenn). The protocol was approved by the Upenn IACUC, and the study adhered to ethical regulations and humane endpoints. Eight-week-old male C57BL / 6N Tac mice were purchased from Taconic Farms, and 8- to 12-week-old male NOD.Cg-Prkdc mice were cultured at 100°C for 12 h. scid Il2rg tm1Wjl (NSG) mice were provided by the Upenn Stem Cell and Xenograft Core.

[0322] Tumor lines and treatments The human acute lymphoblastic leukemia cell line NALM6 and pancreatic adenocarcinoma AsPC1 were obtained from the American Type Culture Collection and authenticated by the University of Arizona Genetics Core. The mouse pancreatic PDA7940b cell line was engineered using the Kras LSL.G12D / + p53 R172H / + (KPC) mouse pancreatic tumor model was established and provided by Gregory Beatty (Upenn).

[0323] NALM6 tumor cells were irradiated with 10,000cGy of X-rays. 1e7 irradiated NALM6 (IR NALM6) cells were injected intravenously into NSG mice every 4 days for a total of 6 injections. C57BL / 6 mice were injected subcutaneously in the back with 1e6 PDA7940b tumor cells in 100μL PBS. NSG mice were injected subcutaneously in the back with 1e6 AsPC1 tumor cells in 100μL 1xMatrigel. Tumor growth was monitored weekly. Tumor volume was determined as length (mm) x width (mm) x 0.5. C57BL / 6 tumor-bearing mice were injected with 1e6 murine CAR-T cells 7 days after tumor inoculation. PDA7940b-bearing C57BL / 6 mice were treated with 1e9 pfu of intratumoral Ad-MCV-mCD19t-eGFP and 200 μg of intraperitoneal αPD-1 (clone 29 F.1A12) monoclonal antibody in 200 μl PBS every other day starting on day 5 after CAR-T injection. NSG tumor-bearing mice were injected with 3e5 or 1e5 CAR-T cells on day 21 after tumor inoculation. 1e7 IR NALM6 cells were injected every 4 days starting on day -1 after CAR-T injection.

[0324] Flow cytometry Single cell suspensions were prepared from liver, tumor, and spleen. Cells were incubated with Fc block (anti-CD16 / 32 clone 93, BioLegend, or Human TruStain FcX™) and stained with the following fluorochrome-conjugated monoclonal antibodies. FMO was used as a negative staining control to set gates. Zombie NIR Fixable Viability Kit and the following fluorochrome-conjugated monoclonal antibodies from BioLegend: 1) Human antibodies: BV605-anti-CD45 (2D1), BV421-anti-PD1 (EH12.2H7), BV510-anti-CD8 (HITa), BV11-anti-CD4 (RPA-T4), BV785-anti-CD3 (OKT3), PE-Cy7-anti-CD27 (M-T271), APC-Cy7-anti-CD39 (A1), Alexa Fluor488-anti-CD45RA (HI100); 2) Mouse antibodies: Alexa Fluor 700-anti-CD45 (30-F11), BV421-anti-PD1 (29F.1A12, RMP1-30), PE-Cy7-anti-CD44 (IM7), BV510-anti-CD45.1 (A20), BV711-anti-CD4 (RM4-5), BV785-anti-CD19 (6D5), BV605-anti-CD8a (53-6.7), PerCP / Cy5.5-anti-Tim3 (B8.2C12), Alexa Fluor 647-anti-CD39 (Duha59), FITC-anti-CD62L (MEL-14), PE / Dazzle™ 594-anti-Lag3 (C9B7W); from ThermoFisher: antibodies for human: PE-eFluor 610-anti-Lag3; and BD Antibodies for humans were purchased from BD Biosciences: Alexa Fluor 700-anti-CD197 (150503). Flow cytometry data were acquired on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0325] The experimental results will now be described.

[0326] Example 1: Design and expression of dual CARs Two dual CAR constructs were designed and each cloned into a pTRPE lentiviral vector, as shown in FIG. 1A. The "pTRPE-hCD19 / 28z-M5BBZ" dual CAR construct encodes a first CAR comprising an anti-human CD19 scFv ("hCD19") and an intracellular domain comprising a human CD28 costimulatory domain ("28") and a human CD3 zeta signaling domain ("z"), followed by a P2A linker, followed by a second CAR comprising an anti-human mesothelin (MSLN) scFv ("M5") and an intracellular domain comprising a human 4-1BB costimulatory domain and a human CD3 zeta signaling domain ("BBZ"), followed by a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). Thus, the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct encodes the hCD19 / 28z CAR upstream (i.e., 5') of the M5BBZ CAR. The "pTRPE-M5BBZ-hCD19 / 28z" dual CAR construct encodes the M5BBZ CAR upstream (i.e., 5') of the hCD19 / 28z CAR, with a P2A linker between the two CARs and a WPRE after the hCD19 / 28z CAR. In both dual CAR constructs, expression is controlled by the constitutive EF1α promoter.

[0327] Additional constructs encoding a single CAR used as controls in the experiments described herein included a pNVS lentiviral construct encoding a single CAR comprising an anti-human CD19 scFv, a human 4-1BB costimulatory domain and a human CD3 zeta signaling domain ("pNVS-hCD19BBZ"), and a pTRPE lentiviral construct encoding a single CAR comprising an anti-human mesothelin (MSLN) scFv ("M5") and an intracellular domain comprising a human 4-1BB costimulatory domain and a human CD3 zeta signaling domain ("pTRPE-M5BBZ").

[0328] The two dual CAR constructs were each transduced into T cells, and the expression of the hCD19 / 28z CAR and M5BBZ CAR was assessed via flow cytometry. As shown in FIG. 1B, T cells containing either dual CAR construct expressed both CARs, but the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct provided higher expression of both CARs compared to the pTRPE-M5BBZ-hCD19 / 28z dual CAR construct.

[0329] A murine version of the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct was also designed and cloned into the MSGV retroviral vector. This murine dual CAR construct ("MSGV-anti-moCD19-MuCD28z-anti-momeso-A03-3-MuBBz") encodes a first CAR comprising an anti-mouse CD19 scFv and a murine CD28 costimulatory domain and a murine CD3 zeta signaling domain, and a second CAR comprising an anti-mouse mesothelin scFv ("A03"), a murine 4-1BB costimulatory domain, and a murine CD3 zeta signaling domain. Control murine CAR constructs include "MSGV-anti-moCD19-MuBBz," which encodes a single CAR comprising an anti-mouse CD19 scFv, a murine 4-1BB costimulatory domain, and a murine CD3 zeta signaling domain, and "MSGV-anti-momeso-A03-3-MuBBz," which encodes a single CAR comprising an anti-mouse mesothelin scFv ("A03"), a murine 4-1BB costimulatory domain, and a murine CD3 zeta signaling domain.

[0330] Example 2: In vitro expansion of dual CAR T cells T cells were transduced with either dual CAR construct (pTRPE-hCD19 / 28z-M5BBZ or pTRPE-M5BBZ-hCD19 / 28z), or with pNVS-hCD19BBZ or pTRPE-M5BBZ single CAR constructs as controls. T cells were co-cultured in vitro for 4 days in the presence and absence of irradiated Nalm6 human tumor cell line. Irradiated Nalm6 expressing human CD19 simulates endogenous CD19+ B cells. Both dual CAR constructs and pNVS-hCD19BBZ showed increased cell numbers of CAR T cells when "boosted" with irradiated NALM6 cells (Figure 2A). However, the pTRPE-hCD19 / 28z-P2A-M5BBZ dual CAR showed less sustained signaling (i.e., less ligand-independent constitutive signaling) in the absence of this CD19 antigen boost. As expected, pTRPE-M5BBz CAR T cell numbers were not boosted by coculture with NALM6 cells (Figure 2A).

[0331] To further evaluate CAR T cell expansion, transduced CAR T cells were labeled with cell trace violet (CTV) and co-cultured with irradiated Nalm6 for 4 days. CD8+ and CD4+ T cells were analyzed for proliferation by division peak. CSFE staining showed similar CAR T expansion for CD4+ and CD8+ T cells transduced with pNVS-hCD19BBZ single CAR construct, pTRPE-M5BBZ-hCD19 / 28z dual CAR construct, or pTRPE-hCD19 / 28z-M5BBz dual CAR construct, but not for pTRPE-M5BBz CAR T cells (Figure 2B).

[0332] Example 3: Boosting dual CAR T cells with CD19 antigen promotes in vitro tumor cell killing We measured in vitro killing of mesothelin-positive AsPC-1 tumor cells by CAR T cells using the xCelligence impedance assay. Cells were inoculated at an effector-to-target ratio (E:T ratio) of 0.1:1 before and after coculture with irradiated CD19+ NALM6 tumor cells (simulating endogenous CD19+ B cells). T cells transduced with either the dual CAR construct or the pTRPE-M5BBZ single CAR construct before boosting with irradiated CD19+ NALM6 tumor cells killed AsPC1-GFP cells equally well in vitro (Figure 3A). Strikingly, after coculture with irradiated CD19+ NALM6 tumor cells, T cells transduced with either dual CAR construct showed enhanced and accelerated tumor cell death (Figure 3B). Interestingly, pTRPE-hCD19 / 28z-M5BBZ dual CAR-T cells exhibited superior (i.e., enhanced) tumor cell death compared to pTRPE-M5BBZ-hCD19 / 28z dual CAR T cells after coculture with irradiated CD19+ NALM6 cells (Figure 3B).

[0333] Example 4: Dual CAR T cells exhibit lower in vitro cytotoxicity against non-tumor normal cells In vitro killing of mesothelin-positive A549 cells by CAR T cells was measured using the xCelligence impedance assay as a model to evaluate the cytotoxic potential of T cells transduced with dual CAR constructs against nontumor normal cells in lung tissue. The cytotoxicity of CAR T cells against A549 cells was measured by the percentage of A549-GFP cell killing over time at E:T=0.1:1. T cells transduced with pTRPE-hCD19 / 28z-M5BBZ dual CAR constructs showed reduced cytotoxicity of A549 cells compared to T cells transduced with pTRPE-M5BBZ-hCD19 / 28z dual CAR or pTRPE-M5BBZ single CAR constructs (Figure 4A and Figure 4B).

[0334] Surprisingly, co-culture of dual CAR cells with irradiated CD19+ Nalm6 cells (simulating endogenous CD19+ B cells) reduced the A549 cytotoxicity of T cells transduced with either dual CAR construct compared to T cells that were not co-cultured with irradiated Nalm6 (Figure 4C). However, T cells transduced with the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct showed a greater reduction in A549 cell cytotoxicity after co-culture with irradiated NALM6 cells compared to T cells transduced with the pTRPE-M5BBZ-CD19 / 28z dual CAR construct (Figure 4C). The increased therapeutic index of T cells transduced with the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct may be because these T cells express lower levels of the M5BBZ CAR compared to T cells transduced with the pTRPE-M5BBZ-hCD19 / 28z dual CAR construct, as indicated by the lower MFI of M5BBZ CAR staining for these cells (Figure 4D).

[0335] Example 5: Boosting dual CAR T cells with CD19 antigen stimulates CAR T expansion in tumor-free NSG mice To assess the expansion of T cells transduced with the dual CAR constructs in tumor-free NSG mice, mice were cultured at 10 7 irradiated Nalm6 cells (simulating endogenous CD19+ B cells) were first administered on day -1, followed by 10 on day 0. 5CAR T cells were administered to mice (Figure 5A). On days 3, 7, 11, 15, and 19, administration of irradiated Nalm6 was repeated, mice were bled on days 3, 11, and 19, and cells were harvested on day 20 (Figure 5A). CAR T cell expansion was assessed in blood samples collected on days 3, 11, and 19. T cells transduced with either the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct or the pTRPE-M5BBZ-hCD19 / 28z dual CAR construct expanded in vivo, where the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct endowed the T cells with superior proliferation capacity compared to the pTRPE-M5BBZ-hCD19 / 28z dual CAR construct (Figure 5B). In contrast, pTRPE-M5BBZ-transduced T cells did not expand (Figure 5C), indicating that stimulation-triggered expansion (i.e., the "boost") was CD19 antigen-driven.

[0336] Next, we evaluated the expansion of CAR T cells in the spleen and liver compared to blood. This showed that the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct significantly enhanced the expansion of T cells compared to T cells transduced with the pTRPE-M5BBZ single CAR construct (Figure 5D). Additionally, a significant increase in the secretion of cytotoxic cytokines (interferon production regulator (IFNr), perforin, granzyme A, and granulysin) in serum was detected for T cells transduced with the pTRPE-hCD19 / 28z-M5BBZ dual CAR construct compared to the pTRPE-M5BBZ single CAR construct (Figure 5E). The liver is the organ where the majority of expanded CAR T cells accumulated in this mouse model, which may be due to the use of irradiated NALM6 tumor lines to boost CAR T expansion. Based on these results, it is expected that in human patients, dual CAR T cell numbers will be boosted by the patient's own CD19+ B cells and thus expand in the blood and induce B cell aplasia, as previously observed with anti-CD19 CAR T cell therapy in human patients.

[0337] The phenotype of cells recovered on day 20 was also evaluated (Figure 5F). Importantly, continuous stimulation of pTRPE-hCD19 / 28z-M5BBZ-transduced T cells with irradiated CD19+ Nalm6 did not result in an exhausted terminally differentiated phenotype. The majority of pTRPE-hCD19 / 28z-M5BBZ CAR T cells still maintained an effector memory cell phenotype, which is similar to T cells transduced with pTRPE-M5BBZ single CAR constructs. Furthermore, the majority of T cells transduced with pTRPE-hCD19 / 28z-M5BBZ dual CAR constructs express only one or two exhaustion markers (PD-1 and / or CD39, but not Tim3 or Lag3). Thus, the observed T cell expansion can be considered as activation rather than exhaustion.

[0338] Example 6: Dual CAR T cells enhance in vivo killing of tumor cells To evaluate the tumor cytotoxicity of T cells transduced with the dual CAR construct in vivo, we inoculated 10 cells into NSG mice. 6 AsPC1 pancreatic tumor cells were initially administered to generate the AsPC1 tumor mouse model, followed by 3 × 10 5 CAR T cells were administered to mice (Figure 6A). Tumor size was assessed periodically for 43 days after T cell injection (Figure 6B and Figure 6C). Even in the absence of administration of irradiated Nalm6 to mice, T cells transduced with the pTRPE-hCD19 / 28z-M5BBZ dual CAR constructs showed significantly enhanced killing of pancreatic tumors in vivo compared to T cells transduced with the pTRPE-M5BBZ single CAR construct (Figure 6B). Administration of irradiated Nalm6 (simulating endogenous CD19+ B cells) further enhanced and promoted the ability of pTRPE-hCD19 / 28z-M5BBZ dual CAR transduced T cells to kill pancreatic tumor cells in vivo (Figure 6C). The enhanced antitumor efficacy of pTRPE-hCD19 / 28z-M5BBZ dual CAR construct-transduced T cells compared to pTRPE-M5BBZ single CAR construct-transduced T cells in the AsPC1 tumor mouse model correlates with higher CAR T cell numbers in the tumor and CAR T cell migration to CD4+ T cells for pTRPE-hCD19 / 28z-M5BBZ dual CAR construct-transduced T cells (Figure 6D). These data demonstrate that CD19 antigen boosts pTRPE-hCD19 / 28z-M5BBZ dual CAR T cells and promotes the antitumor efficacy of these cells.

[0339] Example 7: Mouse dual CAR T cells are similar to human dual CAR T cells In vitro CAR T cell killing of mesothelin-positive PDA7940bWT cell lines was measured using the xCelligence impedance assay. Mouse T cells were transduced with mouse dual CAR constructs (MSGV-anti-moCD19-MuCD28z-anti-momeso-A03-3-MuBBz) or mouse anti-CD19 single CAR constructs or mouse anti-MSLN single CAR constructs as controls. T cells transduced with mouse dual CAR constructs showed enhanced killing of MSLN+ PDA7940bWT cells compared to T cells transduced with anti-MSLN single CAR constructs (Figure 7A).

[0340] We then evaluated the murine dual CAR T cells in a tumor-free syngeneic mouse model. The murine T cells transduced with the murine dual CAR construct, but not either single murine CAR construct, were able to engraft / expand without lymphodepletion in tumor-free syngeneic mice that were depleted of CD19-positive B cells (Figure 7B). The expanded dual CAR T cells were primarily in the spleen compared to the liver (Figure 7C). We analyzed the phenotype of T cells in the blood (Figure 7D) or spleen (Figure 7E) collected on day 7, and these phenotypes were similar to each other. Continuous stimulation of the dual CAR T cells with endogenous CD19+ B cells did not result in an exhausted terminally differentiated phenotype. Approximately 40-50% of the murine dual CAR-T cells were phenotypically central memory cells, which was similar to both of the two murine single CAR-T cells. Overall CAR T cells in the syngeneic model had more central memory cells than CAR T cells in the NSG model with irradiated Nalm6. Additionally, the majority of dual CAR T cells express only one or two exhaustion markers (PD-1 and / or CD39, but not Tim3 or Lag3), and therefore T cell expansion can be considered as activation rather than exhaustion.

[0341] Example 8: Adenoviral delivery of CD19 antigen enhances tumor infiltration of dual CAR T cells To increase CAR T cell trafficking and tumor infiltration, a replication-deficient E1 / E3-deleted adenoviral vector encoding a truncated CD19 antigen fused with eGFP (i.e., Ad-CMV-mCD19t-P2A-eGFP) was administered intratumorally (IT) into C57BL / 6 syngeneic mice after administration of dual CAR T cells (Figure 8). Briefly, PDA (pancreatic) tumors were implanted into C57BL / 6 syngeneic mice on day -7. On day 0, 1e6 CD45.1+ CAR T cells (transduced with either the MSGV-anti-moCD19-MuCD28z-anti-momeso-A03-3-MuBBz dual CAR construct ("19 / 28z-A03") or the MSGV-anti-momeso-A03-3-MuBBz single CAR construct ("A03")) were injected into mice. Mice received alternate-day administrations of 1e9 pfu of Ad-CMV-mCD19t-P2A-eGFP on days 5-11. The negative control group was a CAR transduced with a single anti-mouse mesothelin (A03) CAR. The mice included those that received T cells, those that received null adenoviral vectors, those that did not receive any adenoviral vectors, and combinations thereof. On day 11 after CAR-T injection, tumors were removed and characterized by flow cytometry for expression of CD45.1+ (Figures 9 and 10). A significant increase in infiltration of CAR T cells into tumors was observed in mice that received adenoviral vectors encoding the CD19 antigen (Figure 9), but not in control mice (Figures 9 and 10). Administration of CD19 antigen expressed by adenovirus may have increased TIL infiltration by two mechanisms: inducing tumor infiltration and providing CD19 antigen. Indeed, administration of Ad alone (Ad-null) was insufficient to achieve the same effect (Figure 10). IT injection of Ad-CMV-mCD19t-P2A-eGFP significantly increased CAR-T infiltration, but not CAR T cell infiltration. T cells did not show tumor control and expressed increased levels of PD-1 (Figure 11). The immunosuppressive tumor microenvironment (TME), recapitulated in the syngeneic PDA model, is a known barrier to effective CAR T cell therapy in solid tumors. Therefore, we next combined the treatment regimen with anti-PD1 checkpoint blockade.Administration of anti-PD1 antibody together with CD19 antigen (as Ad-CMV-mCD19t-P2A-eGFP) resulted in reduced tumor size and increased tumor TIL infiltration for mice receiving dual CAR T cells compared to mice receiving CAR T cells expressing only a single A03 CAR (Figure 12 and Figure 13A). As expected, anti-PD1 antibody reduced the percentage and MFI of PD-1 expression in mice receiving dual CAR T cells and CD19 antigen (Figure 13B).

[0342] Mice were then injected with mCAR T cells 7 days after engraftment of "hot" PDA tumors and treated intermittently with adenovirus encoding a truncated CD19 (Figure 14A). Dual mCAR T cells show enhanced tumor burden control (Figure 14B) and overall survival (Figures 14C-14D) against an immunologically "hot" pancreatic ductal adenocarcinoma (PDA), 2838c3, compared with single CAR T cells in the presence and absence of intratumoral treatment with truncated CD19 adenovirus.

[0343] Computational analysis of spleens following "cold" PDA tumor challenge revealed a boost in the spleen (Figures 15A-15B). Applying the same experimental layout described above and shown in Figure 14A using the "cold" tumor PDA model, we characterized CD45.1+ CAR T cells from the spleen by bulk RNA-seq 6 days after CAR T infusion. Data were normalized and analyzed using DESeq2, and the top differentially expressed genes were plotted (Figure 15A). Gene set enrichment analysis was performed utilizing the fGSEA package (Figure 15B). The observed transcriptional changes were consistent with increased activation of T cells and B cell aplasia within the spleen, e.g., downregulation of B cell genes and genes associated with CD4+ T cells in the dual-targeted CAR T cell cohort, as well as GC-B cell interactions. Dual CAR T cells also show high activation scores as demonstrated by upregulation of genes associated with T cell activation, e.g., CD28, Tigit, and PD1 (Figure 15A). This is also complemented by the upregulation of pathways related to proliferation, metabolism, activation, and IFNy signaling without the upregulation of the exhaustion signature (Figure 15B).

[0344] Enumerated Aspects The following enumerated aspects are provided, the numbering of which should not be construed as indicating any level of importance.

[0345] Aspect 1 is (i) a first extracellular antigen-binding domain having affinity for CD19; and (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and A first nucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising: b. (i) a second extracellular antigen-binding domain having affinity for a tumor antigen that is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and A second nucleotide sequence encoding a second CAR comprising: The present invention provides an isolated nucleic acid comprising:

[0346] Embodiment 2 provides the isolated nucleic acid of embodiment 1, wherein the first nucleotide sequence is located 5' to the second nucleotide sequence, or the first nucleotide sequence is located 3' to the second nucleotide sequence.

[0347] Example 3 provides the isolated nucleic acid of Example 1 or Example 2, further comprising a linker nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, the linker nucleotide sequence comprising a ribosomal slippage sequence selected from the group consisting of P2A, T2A, E2A, and F2A.

[0348] Embodiment 4 provides the isolated nucleic acid of any one of the preceding embodiments, further comprising a promoter operably linked to said first nucleotide sequence and / or said second nucleotide sequence.

[0349] Example 5 provides the isolated nucleic acid of Example 4, wherein the promoter is an EF1α promoter.

[0350] Embodiment 6 provides the isolated nucleic acid of any one of the preceding embodiments, further comprising a post-transcriptional regulatory element.

[0351] Embodiment 7 provides the isolated nucleic acid of embodiment 6, wherein the post-transcriptional regulatory element is a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE).

[0352] In an eighth aspect, the tumor antigen is selected from the group consisting of alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DL L3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFRalpha4), HER2, HLA-A2, I CAM1, interleukin-13 receptor subunit alpha (IL3Rα), interleukin-13 receptor subunit alpha 1 (IL13Rα1), interleukin-13 receptor subunit alpha 2 (IL13Rα2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin-4 / FAP, NKG2D-receptor The isolated nucleic acid of any one of the preceding aspects is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-45, IL-45, IL-45, IL-46, IL-47, IL-48, IL-49, IL-5 ...

[0353] Aspect 9 provides the isolated nucleic acid of any one of the preceding aspects, wherein said tumor antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRα4; GFRalpha4), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2).

[0354] Embodiment 10 provides the isolated nucleic acid of any one of the preceding embodiments, wherein the first extracellular antigen binding domain and the second extracellular antigen binding domain are independently selected from a single chain variable fragment (scFv) and a Fab, respectively.

[0355] Example 11 provides the isolated nucleic acid of any one of the preceding examples, wherein at least one costimulatory domain of the first or second intracellular domain comprises a costimulatory domain of a protein independently selected from 4-1BB, CD2, CD28, and ICOS.

[0356] Example 12 provides the isolated nucleic acid of any one of the preceding examples, wherein the signaling domain comprises a CD3 zeta signaling domain.

[0357] Example 13 provides the isolated nucleic acid of any one of the preceding examples, wherein the first and / or second transmembrane domain comprises a transmembrane domain of a protein independently selected from CD8, CD28, and 4-1BB.

[0358] Embodiment 14 provides the isolated nucleic acid of any one of the preceding embodiments, wherein said first and / or second CAR further comprises a hinge domain.

[0359] Example 15 provides the isolated nucleic acid of Example 14, wherein the hinge domain comprises a CD8 hinge domain.

[0360] Aspect 16 is a method for producing a a. the first CAR comprises: (i) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Includes; b. the second CAR comprises: (i) a second extracellular antigen-binding domain comprising an anti-tumor antigen scFv, wherein the tumor antigen is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Including, An isolated nucleic acid of any one of the preceding aspects is provided.

[0361] Aspect 17 is a method for producing a a. the first CAR comprises: (i) the CD8 leader sequence; (ii) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; and (iii) CD8 hinge and; (iv) a CD28 transmembrane domain; and (v) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and consists essentially of; b. the second CAR comprises: (i) the CD8 leader sequence; (ii) a second extracellular antigen-binding domain comprising an anti-mesothelin scFv; and (iii) CD8 hinge and; (iv) a CD8 transmembrane domain; and (v) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and consisting essentially of An isolated nucleic acid of any one of the preceding aspects is provided.

[0362] Embodiment 18 further comprises a third nucleotide sequence encoding a switch receptor, the switch receptor comprising an extracellular domain of a first receptor and an intracellular domain of a second receptor, the first receptor and the second receptor being, respectively, TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, TIM3 and and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R.

[0363] Example 19 provides the isolated nucleic acid of any one of the preceding examples, further comprising a fourth nucleotide sequence encoding a dominant negative receptor that is a dnTGFβR.

[0364] Embodiment 20 provides a vector comprising the isolated nucleic acid of any one of the preceding embodiments.

[0365] Embodiment 21 provides the vector of embodiment 26, which is a lentiviral vector or a retroviral vector.

[0366] Embodiment 22 provides a modified cell comprising the isolated nucleic acid of any one of embodiments 1-19 or the vector of any one of embodiments 20-21.

[0367] Embodiment 23 provides the modified cell of embodiment 22, wherein the modified cell is selected from a bacterial cell, a fungal cell, a yeast cell, an insect cell, an animal cell, a mammalian cell, and a human cell.

[0368] Embodiment 24 provides the modified cell of embodiment 23, which is a mammalian or human cell, and further which is an immune cell or a precursor cell thereof.

[0369] Example 25 provides the modified cell of Example 24, wherein the immune cell is a T cell.

[0370] Aspect 26 is A modified cell that is an immune cell or a precursor cell thereof. (i) a first extracellular antigen-binding domain having affinity for CD19; and (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and A first chimeric antigen receptor (CAR) comprising: b. (i) a second extracellular antigen-binding domain having affinity for a tumor antigen that is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and The second CAR containing The modified cell is engineered to express

[0371] Aspect 27 is directed to a method for treating cancer, the method comprising administering to a patient a tumor antigen selected from the group consisting of alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, 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, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin-13 receptor subunit alpha (IL3Rα), interleukin-13 receptor subunit nit alpha 1 (IL13Rα1), interleukin 13 receptor subunit alpha 2 (IL13Rα2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin 4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1–6), New York esophageal squamous cell carcinoma-1 (NY-ESO 27. The modified cell of embodiment 26, wherein the modified cell is selected from the group consisting of: PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4; GFR alpha4), VEGFR2, and any combination thereof.

[0372] Example 28 provides the modified cell of example 26 or example 27, wherein said tumor antigen is selected from the group consisting of prostate specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), glycosyl-phosphatidylinositol (GPI)-linked GDNF family alpha-receptor 4 (GFRα4; GFRalpha4), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), and interleukin 13 receptor subunit alpha 2 (IL13Rα2).

[0373] Example 29 provides the modified cell of any one of the preceding examples, wherein the first extracellular antigen binding domain and the second extracellular antigen binding domain are independently selected from a single chain variable fragment (scFv) and a Fab, respectively.

[0374] Example 30 provides the modified cell of any one of the preceding examples, wherein at least one costimulatory domain of the first or second intracellular domain comprises a costimulatory domain of a protein independently selected from 4-1BB, CD2, CD28, and ICOS.

[0375] Example 31 provides the modified cell of any one of the preceding examples, wherein the signaling domain comprises a CD3 zeta signaling domain.

[0376] Example 32 provides the modified cell of any one of the preceding examples, wherein the first and / or second transmembrane domain comprises a transmembrane domain of a protein independently selected from CD8, CD28, and 4-1BB.

[0377] Embodiment 33 provides the modified cell of any one of the preceding embodiments, wherein said first and / or second CAR further comprises a hinge domain.

[0378] Embodiment 34 provides the modified cell of embodiment 33, wherein the hinge domain comprises a CD8 hinge domain.

[0379] Aspect 35 is a. the first CAR comprises: (i) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Includes; b. the second CAR comprises: (i) a second extracellular antigen-binding domain comprising an anti-tumor antigen scFv, wherein the tumor antigen is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Including, The modified cell of any one of the preceding aspects is provided.

[0380] Aspect 36 is a. the first CAR comprises: (i) the CD8 leader sequence; (ii) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; and (iii) CD8 hinge and; (iv) a CD28 transmembrane domain; and (v) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and consists essentially of; b. the second CAR comprises: (i) the CD8 leader sequence; (ii) a second extracellular antigen-binding domain comprising an anti-mesothelin scFv; and (iii) CD8 hinge and; (iv) a CD8 transmembrane domain; and (v) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and consisting essentially of The modified cell of any one of the preceding aspects is provided.

[0381] Embodiment 37 is further engineered to express a switch receptor, the switch receptor comprising an extracellular domain of a first receptor and an intracellular domain of a second receptor, the first receptor and the second receptor being, respectively, TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, TIM3 and and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R.

[0382] Example 38 provides the modified cell of any one of the preceding examples, further engineered to express a dominant negative receptor that is a dnTGFβR.

[0383] Embodiment 39 provides the modified cell of any one of the preceding embodiments, comprising a vector encoding said first CAR and said second CAR.

[0384] Example 40 provides the modified cell of Example 39, wherein said vector is a lentiviral vector or a retroviral vector.

[0385] Example 41 provides the modified cell of example 39 or example 40, wherein said vector further encodes the switch receptor of example 37 and / or the dominant negative receptor of example 38.

[0386] Example 42 provides the modified cell of any one of the preceding examples, which is a mouse cell or a human cell.

[0387] Example 43 provides the modified cell of any one of the preceding examples, which is a T cell.

[0388] Example 44 provides a pharmaceutical composition comprising a population of modified cells of any one of the preceding examples and at least one pharma- ceutically acceptable carrier.

[0389] Aspect 45 is A method of treating cancer in a subject in need thereof is provided, comprising administering to the subject the pharmaceutical composition of embodiment 43.

[0390] Aspect 46 is 1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a population of modified cells, the cells being immune cells or precursor cells thereof, the cells comprising: (i) a first extracellular antigen-binding domain having affinity for CD19; and (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and A first chimeric antigen receptor (CAR) comprising: b. (i) a second extracellular antigen-binding domain having affinity for a tumor antigen that is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and The second CAR containing The method further comprises the step of:

[0391] Aspect 47 is directed to a method for treating cancer, the method comprising administering to a patient a tumor antigen selected from the group consisting of alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, and claudin 1. 8.2, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin-13 receptor subunit alpha (IL3Ra ), interleukin-13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin-4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1-6), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16 , PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4; GFR alpha4), VEGFR2, and any combination thereof.

[0392] Example 48 provides the method of example 46 or example 47, wherein said tumor antigen is selected from the group consisting of prostate specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), glycosyl-phosphatidylinositol (GPI)-linked GDNF family alpha-receptor 4 (GFRα4; GFRalpha4), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2).

[0393] Embodiment 49 provides the method of any one of the preceding embodiments, wherein the first extracellular antigen binding domain and the second extracellular antigen binding domain are independently selected from a single chain variable fragment (scFv) and a Fab, respectively.

[0394] Embodiment 50 provides the method of any one of the preceding embodiments, wherein at least one costimulatory domain of the first or second intracellular domain comprises a costimulatory domain of a protein independently selected from 4-1BB, CD2, CD28, and ICOS.

[0395] Embodiment 51 provides the method of any one of the preceding embodiments, wherein the signaling domain comprises a CD3 zeta signaling domain.

[0396] Example 52 provides the method of any one of the preceding examples, wherein the first and / or second transmembrane domain comprises a transmembrane domain of a protein independently selected from CD8, CD28, and 4-1BB.

[0397] Embodiment 53 provides the method of any one of the preceding embodiments, wherein said first and / or second CAR further comprises a hinge domain.

[0398] Embodiment 54 provides the method of embodiment 53, wherein the hinge domain comprises a CD8 hinge domain.

[0399] Aspect 55 is a method for producing a a. the first CAR comprises: (i) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Includes; b. the second CAR comprises: (i) a second extracellular antigen-binding domain comprising an anti-tumor antigen scFv, wherein the tumor antigen is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Including, The method of any one of the preceding aspects is provided.

[0400] Aspect 56 is a method for producing a a. the first CAR comprises: (i) the CD8 leader sequence; (ii) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; and (iii) CD8 hinge and; (iv) a CD28 transmembrane domain; and (v) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and consists essentially of; b. the second CAR comprises: (i) the CD8 leader sequence; (ii) a second extracellular antigen-binding domain comprising an anti-mesothelin scFv; and (iii) CD8 hinge and; (iv) a CD8 transmembrane domain; and (v) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and consisting essentially of The method of any one of the preceding aspects is provided.

[0401]

[0023] Embodiment 57 is a method for producing a modified cell comprising the steps of: (a) expressing a switch receptor, the switch receptor comprising an extracellular domain of a first receptor and an intracellular domain of a second receptor, the first receptor and the second receptor being selected from the group consisting of TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, T The method of any one of the preceding aspects is provided, wherein the IFNγ-binding domain is selected from the group consisting of IFNγ and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R.

[0402] Example 58 provides the method of any one of the preceding examples, wherein the modified cells are further engineered to express a dominant negative receptor that is a dnTGFβR.

[0403] Embodiment 59 provides the method of any one of the preceding embodiments, wherein the modified cell comprises a vector encoding the first CAR and the second CAR.

[0404] Example 60 provides the method of example 59, wherein the vector is a lentiviral vector or a retroviral vector.

[0405] Example 61 provides the method of example 59 or example 60, wherein said vector further encodes the switch receptor of example 57 and / or the dominant negative receptor of example 58.

[0406] Example 62 provides the method of any one of the preceding examples, wherein the modified cell is a mouse cell or a human cell.

[0407] Example 63 provides the method of any one of the preceding examples, wherein the population of modified cells comprises T cells.

[0408] Example 64 provides the method of any one of the preceding examples, wherein the modified cells are autologous to the subject.

[0409] Example 65 provides the method of any one of the preceding examples, wherein the modified cells are allogeneic to the subject.

[0410] Example 66 provides the method of any one of the preceding examples, wherein the population of modified cells is administered as a pharmaceutical composition comprising the population of modified cells and at least one pharma- ceutically acceptable carrier.

[0411] Aspect 67 is a method for determining whether the population of modified cells is about 1 x 10 6 ~Approx. 1×10 9 The method of any one of the preceding aspects is provided, comprising a cell.

[0412] Example 68 provides the method of any one of the preceding examples, wherein the modified cells exhibit expansion in peripheral blood of the subject.

[0413] Example 69 provides the method of example 68, wherein said expansion is at least 10 fold, at least 100 fold, or at least 1000 fold.

[0414] Embodiment 70 provides the method of any one of the preceding embodiments, wherein the modified cells are detectable for at least 24 months after administering the cells.

[0415] Example 71 provides the method of any one of the preceding examples, wherein the subject is a human.

[0416] Example 72 provides the method of any one of the preceding examples, wherein the cancer is selected from breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, prostate cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid cancer.

[0417]

[0036] Embodiment 73 provides the method of any one of the preceding embodiments, wherein the population of modified cells comprises T cells, and at least 30% or at least 40% of the population of modified cells are phenotypically central memory T cells at or after day 7 post-administration.

[0418] Embodiment 74 provides the method of any one of the preceding embodiments, further comprising administering a CD19 antigen to the subject.

[0419] Embodiment 75 provides the method of embodiment 74, wherein the step of administering the CD19 antigen comprises administering a vector encoding the CD19 antigen or cells engineered to express the CD19 antigen.

[0420] Embodiment 76 provides the method of embodiment 74 or 75, wherein the CD19 antigen comprises the CD19 extracellular domain or an antigenic fragment thereof.

[0421] Example 77 provides the method of any one of examples 74 to 76, wherein a CD19 antigen is administered prior to, simultaneously with, or following administration of said population of modified cells.

[0422] Example 78 provides the method of any one of Examples 75 to 77, wherein the vector is an adenoviral vector.

[0423] Example 79 provides the method of any one of Examples 74 to 78, further comprising administering to the subject an anti-PD1 immunotherapy.

[0424] Embodiment 80 provides the method of embodiment 79, wherein the anti-PD1 immunotherapy is an anti-PD1 antibody.

[0425] Other Aspects The disclosures of any and all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and modifications of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent modifications.

Claims

1. a. (i) a first extracellular antigen-binding domain having affinity for CD19; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and a first nucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising: b. (i) a second extracellular antigen-binding domain having affinity for a tumor antigen that is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and A second nucleotide sequence encoding a second CAR comprising: An isolated nucleic acid comprising: (a) the first nucleotide sequence is located 5' to the second nucleotide sequence; and / or (b) the isolated nucleic acid further comprises a linker nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, the linker nucleotide sequence comprising a ribosomal slippage sequence selected from the group consisting of P2A, T2A, E2A, and F2A; and / or (c) the isolated nucleic acid further comprises a promoter operably linked to the first nucleotide sequence and / or the second nucleotide sequence, optionally wherein the promoter is an EF1α promoter; and / or (d) the isolated nucleic acid further comprises a post-transcriptional regulatory element, optionally the post-transcriptional regulatory element is a woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE); and / or (e) the tumor antigen is alpha-fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, 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, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4; GFRalpha4), HER2, and / or selected from the group consisting of HLA-A2, ICAM1, interleukin-13 receptor subunit alpha (IL3Rα), interleukin-13 receptor subunit alpha 1 (IL13Rα1), interleukin-13 receptor subunit alpha 2 (IL13Rα2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin-4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1-6), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), VEGFR2, and any combination thereof; and / or (f) the tumor antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4; GFRalpha4), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2); and / or (g) the first extracellular antigen-binding domain and the second extracellular antigen-binding domain are independently selected from the group consisting of a single-chain variable fragment (scFv) and a Fab; and / or (h) at least one costimulatory domain of the first or second intracellular domain comprises a costimulatory domain of a protein independently selected from the group consisting of 4-1BB, CD2, CD28, and ICOS; and / or (i) the signaling domain comprises a CD3 zeta signaling domain; and / or (j) the first and / or second transmembrane domains comprise transmembrane domains of proteins independently selected from the group consisting of CD8, CD28, and 4-1BB; and / or (k) the first and / or second CAR further comprises a hinge domain, optionally, the hinge domain comprises a CD8 hinge domain; and / or (l) the isolated nucleic acid further comprises a nucleotide sequence encoding a switch receptor, the switch receptor comprising the extracellular domain of a first receptor and the intracellular domain of a second receptor, the first receptor and the second receptor being selected from the group consisting of TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, TIM3, and and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, VISTA and IL-9R, TIGIT and IL-9R, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R; and / or (m) the isolated nucleic acid further comprises a nucleotide sequence encoding a dominant-negative receptor that is a dnTGFβR; 2. The isolated nucleic acid of claim 1.

3. a. the first CAR is: (i) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Including; b. the second CAR: (i) a second extracellular antigen-binding domain comprising an anti-tumor antigen scFv, wherein the tumor antigen is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Contains, or a'. the first CAR is (i) a CD8 leader sequence; (ii) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (iii) CD8 hinge and; (iv) a CD28 transmembrane domain; and (v) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Including; b'. the second CAR is (i) a CD8 leader sequence; (ii) a second extracellular antigen-binding domain comprising an anti-mesothelin scFv; and (iii) CD8 hinge and; (iv) a CD8 transmembrane domain; and (v) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Including, 2. The isolated nucleic acid of claim 1.

4. A vector comprising the isolated nucleic acid of any one of claims 1 to 3, and optionally being a lentiviral vector or a retroviral vector.

5. 4. A modified cell comprising the isolated nucleic acid of any one of claims 1 to 3, and optionally selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an insect cell, an animal cell, a mammalian cell, and a human cell, and further optionally being a mammalian cell or a human cell, and further optionally being an immune cell or a precursor cell thereof, and further optionally wherein the immune cell is a T cell.

6. A modified cell that is an immune cell or a precursor cell thereof. a. (i) a first extracellular antigen-binding domain having affinity for CD19; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and a first chimeric antigen receptor (CAR) comprising: b. (i) a second extracellular antigen-binding domain having affinity for a tumor antigen that is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and The second CAR containing The modified cell is engineered to express

7. (a) The tumor antigen is alpha-fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, or c-Met. , DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin-13 receptor subunit alpha (IL3Rα), interleukin-13 receptor subunit subunit alpha 1 (IL13Rα1), interleukin-13 receptor subunit alpha 2 (IL13Rα2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin-4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1-6), New York esophageal squamous cell carcinoma-1 (NY -ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4; GFR alpha 4), VEGFR2, and any combination thereof; and / or (b) the tumor antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4; GFRalpha4), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), and interleukin-13 receptor subunit alpha 2 (IL13Rα2); and / or (c) the first extracellular antigen-binding domain and the second extracellular antigen-binding domain are independently selected from the group consisting of a single-chain variable fragment (scFv) and a Fab; and / or (d) at least one costimulatory domain of the first or second intracellular domain comprises a costimulatory domain of a protein independently selected from the group consisting of 4-1BB, CD2, CD28, and ICOS; and / or (e) the signaling domain comprises a CD3 zeta signaling domain; and / or (f) the first and / or second transmembrane domains comprise transmembrane domains of proteins independently selected from the group consisting of CD8, CD28, and 4-1BB; and / or (g) the first and / or second CAR further comprises a hinge domain, optionally, the hinge domain comprises a CD8 hinge domain; and / or (h) the cell is further engineered to express a switch receptor, the switch receptor comprising the extracellular domain of a first receptor and the intracellular domain of a second receptor, the first receptor and the second receptor being selected from the group consisting of TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, and TIM3 and IL1 2R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, VISTA and IL-9R, TIGIT and IL-9R, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R; and / or (i) the cells have been further engineered to express a dominant-negative receptor that is a dnTGFβR, and / or (j) the cell comprises a vector encoding the first CAR and the second CAR, optionally wherein the vector is a lentiviral vector or a retroviral vector, and optionally wherein the vector further encodes (i) a switch receptor, wherein the switch receptor comprises the extracellular domain of a first receptor and the intracellular domain of a second receptor, and wherein the first receptor and the second receptor are selected from the group consisting of TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, and CTLA4, respectively. and CD28, TIM3 and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD 28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, VISTA and IL-9R, TIGIT and IL-9R, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R, and / or (ii) a dominant-negative receptor which is a dnTGFβR; and / or (k) the cell is a mouse cell or a human cell; and / or (l) the cell is a T cell; The modified cell of claim 6.

8. a. the first CAR is: (i) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Including; b. the second CAR: (i) a second extracellular antigen-binding domain comprising an anti-tumor antigen scFv, wherein the tumor antigen is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Contains, or a'. the first CAR is (i) a CD8 leader sequence; (ii) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (iii) CD8 hinge and; (iv) a CD28 transmembrane domain; and (v) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Including; b'. the second CAR is (i) a CD8 leader sequence; (ii) a second extracellular antigen-binding domain comprising an anti-mesothelin scFv; and (iii) CD8 hinge and; (iv) a CD8 transmembrane domain; and (v) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Including, The modified cell of claim 6.

9. A pharmaceutical composition comprising the population of modified cells of any one of claims 6 to 8 and at least one pharmaceutically acceptable carrier.

10. 10. The pharmaceutical composition of claim 9, for use in a method for treating cancer in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the pharmaceutical composition.

11. 1. A pharmaceutical composition comprising a population of modified cells for use in a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject a population of modified cells, the population of modified cells being immune cells or precursor cells thereof, the modified cells comprising: a. (i) a first extracellular antigen-binding domain having affinity for CD19; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising at least one costimulatory domain and a signaling domain; and a first chimeric antigen receptor (CAR) comprising: b. (i) a second extracellular antigen-binding domain having affinity for a tumor antigen that is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising at least one costimulatory domain and a signaling domain; and The second CAR containing The pharmaceutical composition is engineered to express

12. (a) The tumor antigen is alpha-fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, or claudin 18 .2, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin-13 receptor subunit alpha (IL3R a), interleukin-13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin-4 / FAP, NKG2D-ligands (MIC-A, MIC-B, and ULBP1-6), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, ROR2, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUC1), glycosyl-phosphatidylinositol (GPI)-linked GDNF family alpha-receptor 4 (GFRα4; GFRalpha4), VEGFR2, and any combination thereof; and / or (b) the tumor antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), MUC1, Tn-glycoform of MUC1 (TnMUC1), folate receptor alpha (FRα), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glypican 2 (GPC2), glycosyl-phosphatidylinositol (GPI)-linked GDNF family α-receptor 4 (GFRα4; GFRalpha4), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 1 (IL13Rα1), and interleukin-13 receptor subunit alpha 2 (IL13Rα2); and / or (c) the first extracellular antigen-binding domain and the second extracellular antigen-binding domain are independently selected from the group consisting of a single-chain variable fragment (scFv) and a Fab; and / or (d) at least one costimulatory domain of the first or second intracellular domain comprises a costimulatory domain of a protein independently selected from the group consisting of 4-1BB, CD2, CD28, and ICOS; and / or (e) the signaling domain comprises a CD3 zeta signaling domain; and / or (f) the first and / or second transmembrane domains comprise transmembrane domains of proteins independently selected from the group consisting of CD8, CD28, and 4-1BB; and / or (g) the first and / or second CAR further comprises a hinge domain, optionally, the hinge domain comprises a CD8 hinge domain; and / or (h) the modified cell is further engineered to express a switch receptor, the switch receptor comprising the extracellular domain of a first receptor and the intracellular domain of a second receptor, the first receptor and the second receptor being selected from the group consisting of TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, TIM3 and IL 12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3-IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, VISTA and IL-9R, TIGIT and IL-9R, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R; and / or (i) the modified cells are further engineered to express a dominant-negative receptor that is a dnTGFβR; and / or (j) the modified cell comprises a vector encoding the first CAR and the second CAR, optionally wherein the vector is a lentiviral vector or a retroviral vector, and optionally wherein the vector further encodes (i) a switch receptor, wherein the switch receptor comprises the extracellular domain of a first receptor and the intracellular domain of a second receptor, and wherein the first receptor and the second receptor are, respectively, TGFβR and IL12R, TGFβR and CD28, TGFβR and OX40, TGFβR and CD27, TGFβR and 4-1BB, TGFβR and IL-2R, TGFβR and IL-9R, PD1 and IL12R, PD1 and CD28, PD1 and ICOS, PD1 and CD27, PD1 and 4-1BB, PD1 and IL-2R, PD1 and IL-9R, BTLA and CD28, BTLA and ICOS, BTLA and CD27, CTLA4 and CD28, TIM3 and IL12R, TIM3 and CD28, TIM3 and CD28, TIM3 and OX40, TIM3 and 4-1BB, TIM3 and IL-2R, TIM3 and IL-9R, VSIG3 and CD28, VSIG3-IL12Rβ2, VSIG3 and CD27, VSIG3 and 4-1BB, VSIG3 and ICOS, VSIG3- further encoding a dominant-negative receptor selected from the group consisting of IL12Rβ1, VSIG8-IL12Rβ1, VSIG8 and CD28, VSIG8-IL12Rβ2, VSIG8 and CD27, VSIG8 and 4-1BB, VSIG8-ICOS, VISTA and IL-9R, TIGIT and IL-9R, IFNγ and CD28, IFNγ and OX40, IFNγ and IL2R, and IFNγ and IL12R, and / or (ii) a dnTGFβR; and / or (k) the modified cell is a human cell; and / or (l) the population of modified cells comprises T cells; and / or (m) the modified cells are autologous to the human subject; and / or (n) the modified cells are allogeneic to the human subject; and / or (o) the population of modified cells is administered as a pharmaceutical composition comprising the population of modified cells and at least one pharmaceutically acceptable carrier; and / or (p) the population of modified cells comprises about 1×10 6 to about 1×10 9 cells, and optionally, the modified cells exhibit expansion in the peripheral blood of the human subject; and / or (q) the expansion is at least 10-fold, at least 100-fold, or at least 1000-fold; and / or (r) the modified cells are detectable for at least 24 months after administration of the cells; and / or (s) the cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid cancer; and / or (t) the population of modified cells comprises T cells, and at least 30% or at least 40% of the population of modified cells are phenotypically central memory T cells at or after day 7 after administration; and / or (u) the method further comprises administering a CD19 antigen to the human subject, and optionally (i) the step of administering the CD19 antigen comprises administering a vector encoding the CD19 antigen or cells engineered to express the CD19 antigen, optionally wherein the vector is an adenoviral vector; and / or (ii) the CD19 antigen comprises the CD19 extracellular domain or an antigenic fragment thereof, and / or (iii) a CD19 antigen is administered prior to, simultaneously with, or after administration of the population of modified cells; and / or (iv) the method further comprises administering to the subject an anti-PD1 immunotherapy, optionally wherein the anti-PD1 immunotherapy is an anti-PD1 antibody; 12. The pharmaceutical composition of claim 11.

13. a. the first CAR is: (i) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (ii) a first transmembrane domain; and (iii) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Including; b. the second CAR: (i) a second extracellular antigen-binding domain comprising an anti-tumor antigen scFv, wherein the tumor antigen is not CD19; and (ii) a second transmembrane domain; and (iii) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Contains, or a'. the first CAR is (i) a CD8 leader sequence; (ii) a first extracellular antigen-binding domain comprising an anti-CD19 scFv; (iii) CD8 hinge and; (iv) a CD28 transmembrane domain; and (v) a first intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta signaling domain; and Including; b. the second CAR: (i) a CD8 leader sequence; (ii) a second extracellular antigen-binding domain comprising an anti-mesothelin scFv; and (iii) CD8 hinge and; (iv) a CD8 transmembrane domain; and (v) a second intracellular domain comprising a 4-1BB costimulatory domain and a CD3 zeta signaling domain; and Including, 13. The pharmaceutical composition of claim 11 or 12.

14. (a) (i) a T cell comprising the nucleic acid of claim 1 or claim 3, and (ii) a vector encoding or a cell engineered to express the CD19 antigen; or (b) (i) a T cell comprising the nucleic acid of claim 1 or claim 3, and (ii) an anti-PD1 antibody; or (c) (i) a T cell comprising the nucleic acid of claim 1 or claim 3, (ii) a vector encoding the CD19 antigen or a cell engineered to express the CD19 antigen, and (iii) an anti-PD1 antibody. A medicine comprising a combination of the above.

15. A pharmaceutical agent as described in claim 14 for use in a method of treating cancer in a human subject in need thereof, the method comprising the step of administering an effective amount of the pharmaceutical agent to the human subject, and optionally the cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid cancer.

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