SPLIT RECEPTOR SWITCH POLYPEPTIDES AND USES THEREOF

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/419,096, filed October 25, 2022, which application is incorporated herein by reference in its entirety.

BACKGOUND OF THE DISCLOSURE

[0002] Immunotherapeutic CAR-T cell (chimeric antigen receptor T cell) therapies have been used for the treatment of diseases such as B-cell lymphoma or for patients who have relapsed after receiving chemotherapy. One of the important criteria for a successful CAR-T cell therapy is robust ex vivo expansion of the CAR-T cells before infusion. Cytokines are often used for expanding T cells, such as IL-2. However, using these cytokines would expand both interested CAR-T cells and native T cells, resulting in low yield and low purity of the interested CAR-T cells in the final cell population that will be infused to the patients.

[0003] Therefore, there remains an urgent medical need for a robust selective expansion of the desired CAR-T cells to result a high yield and high purity of the CAR-T cells in the cell population.

SUMMARY OF THE DISCLOSURE

[0004] Disclosed herein is a composition comprising a recombinant polynucleic acid comprising a first sequence encoding a first polypeptide comprising a first portion of a split receptor switch polypeptide and a second sequence encoding a second polypeptide comprising a second portion of the split receptor switch polypeptide, wherein the first polypeptide comprises: (a) a first extracellular domain comprising a cytokine binding domain from a first cytokine receptor, and (b) a first transmembrane domain, wherein the second polypeptide comprises: (a) a second transmembrane domain, and (b) a first intracellular domain comprising an intracellular signaling domain from a second cytokine receptor; wherein the first cytokine receptor and the second cytokine receptor are different cytokine receptors.

[0005] In some embodiments, the first cytokine receptor is an IL9 or IL21 receptor. In some embodiments, the first cytokine receptor is an IL-2RG (CD 132) receptor. In some embodiments, the first polypeptide and the second polypeptide form a complex comprising the split receptor switch polypeptide. In some embodiments, the first polypeptide and the second polypeptide form a complex. In some embodiments, the split receptor switch polypeptide is a functional split receptor switch polypeptide. [0006] In some embodiments, the first transmembrane domain interacts with the second transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain form a multimer. In some embodiments, the first transmembrane domain and the second transmembrane domain form a homodimer or a heterodimer. In some embodiments, the second polypeptide is not a functional split receptor switch polypeptide. In some embodiments, the split receptor switch polypeptide is expressed in a cell. In some embodiments, the split receptor switch polypeptide is a functional split receptor switch polypeptide capable of signaling to the cell. In some embodiments, the first polypeptide is not a functional split receptor switch polypeptide capable of signaling to the cell. In some embodiments, the second polypeptide is not a functional split receptor switch polypeptide capable of signaling to the cell.

[0007] In some embodiments, the first intracellular signaling domain is activated in the presence of a cytokine that binds to the extracellular domain of the first polypeptide. In some embodiments, the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide in a cell that expresses both the first and second polypeptide compared to a cell that expresses only the first polypeptide, only the second polypeptide or neither the first polypeptide nor the second polypeptide. In some embodiments, the cell expands at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that expresses only the first polypeptide, only the second polypeptide, or neither the first polypeptide nor the second polypeptide.

[0008] In some embodiments, the cell expresses endogenous IL-2RG (CD 132) receptor. In some embodiments, the endogenous IL-2RG (CD 132) receptor forms a complex with the first polypeptide, the second polypeptide or both. In some embodiments, the first polypeptide, the second polypeptide and the endogenous IL-2RG (CD 132) receptor form a complex comprising the split receptor switch polypeptide. In some embodiments, the first polypeptide, the second polypeptide and the endogenous IL-2RG (CD 132) receptor form a complex comprising the split receptor switch polypeptide in the presence of a cytokine. In some embodiments, the first polypeptide, the second polypeptide and the endogenous IL-2RG (CD 132) receptor do not form a complex comprising the split receptor switch polypeptide in the absence of a cytokine. In some embodiments, the cell does not express endogenous IL-2RG (CD 132) receptor. In some embodiments, the cell expresses an exogenous IL-2RG (CD 132). In some embodiments, the composition further comprises a recombinant polynucleic acid comprising a sequence encoding IL-2RG (CD 132). In some embodiments, the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that does not express a IL-2RG (CD 132) receptor. [0009] In some embodiments, the first polypeptide further comprises an intracellular domain from a third cytokine receptor. In some embodiments, the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor and the second cytokine receptor. In some embodiments, the second polypeptide further comprises a second extracellular domain from a third cytokine receptor. In some embodiments, the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor and the second cytokine receptor. In some embodiments, the first extracellular domain of the first polypeptide comprises a first portion of the cytokine binding domain, and the second extracellular domain of the third polypeptide comprises a second portion of the cytokine binding domain. In some embodiments, the third cytokine receptor is an IL-2RG (CD 132) receptor.

[0010] In some embodiments, the recombinant polynucleic acid comprises a first recombinant polynucleic acid molecule comprising the first sequence and a second recombinant polynucleic acid molecule comprising the second sequence. In some embodiments, the first recombinant polynucleic acid further comprises a sequence encoding a first gene of interest. In some embodiments, the sequence encoding a first gene of interest gene of interest encodes for a first protein of interest, wherein the first polypeptide and the first protein of interest are expressed as a single polypeptide molecule. In some embodiments, the sequence encoding the first polypeptide is linked to the encoding the first protein of interest by a sequence encoding a linker. In some embodiments, the linker comprises a self-cleaving peptide sequence, a protease cleavage site or an IRES.

[0011] In some embodiments, the second recombinant polynucleic acid further comprises a sequence encoding a second gene of interest. In some embodiments, the sequence encoding a second gene of interest gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as a single polypeptide molecule. In some embodiments, the sequence encoding the second polypeptide is linked to the encoding the second protein of interest by a sequence encoding a linker. In some embodiments, the composition further comprises a third recombinant polynucleic acid comprising a sequence encoding a third polypeptide. In some embodiments, the first sequence and the second sequence are not comprised within the same recombinant polynucleic acid molecule. In some embodiments, the recombinant polynucleic acid further comprises a third sequence encoding a third polypeptide comprising a third portion of the split receptor switch polypeptide. In some embodiments, the third recombinant polynucleic acid further comprises a sequence encoding a third gene of interest. In some embodiments, the sequence encoding a third gene of interest encodes for a third protein of interest, wherein the third polypeptide and the third protein of interest are expressed as a single polypeptide molecule. In some embodiments, the sequence encoding the second polypeptide is linked to the encoding the second protein of interest by a sequence encoding a linker. In some embodiments, the linker comprises a self-cleaving peptide sequence, a protease cleavage site or an IRES.

[0012] In some embodiments, the third polypeptide comprises: a third transmembrane domain, and a second intracellular domain comprising an intracellular domain from a third cytokine receptor. In some embodiments, the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor. In some embodiments, the third cytokine receptor is a cytokine receptor that is different than the second cytokine receptor. In some embodiments, the third polypeptide comprises: a second extracellular domain from a third cytokine receptor; and a third transmembrane domain. In some embodiments, the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor. In some embodiments, the third cytokine receptor is a cytokine receptor that is different than the second cytokine receptor. In some embodiments, the third polypeptide comprises: a second extracellular domain from a third cytokine receptor, wherein the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor and the second cytokine receptor; a third transmembrane domain; and a second intracellular domain comprising an intracellular domain from the first cytokine receptor. In some embodiments, the third cytokine receptor of the third polypeptide is IL-2RG (CD 132). In some embodiments, the first cytokine receptor of the third polypeptide is IL- 2RG.

[0013] In some embodiments, the third transmembrane domain interacts with the first transmembrane domain and/or the second transmembrane domain. In some embodiments, the first transmembrane domain, the second transmembrane domain and the third transmembrane domain form a multimer. In some embodiments, the third transmembrane domain interacts with the first transmembrane domain but not the second transmembrane domain. In some embodiments, the first transmembrane domain and the third transmembrane domain form a multimer. In some embodiments, the third transmembrane domain interacts with the second transmembrane domain but not the first transmembrane domain. In some embodiments, the second transmembrane domain and the third transmembrane domain form a multimer. In some embodiments, the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide in a cell that expresses the first, second and third polypeptide compared to a cell that expresses the first polypeptide, the second polypeptide, the third polypeptide, the first and second polypeptide, the first and third polypeptide, the second and third polypeptide or none of the first, second and third polypeptides. In some embodiments, the cell that expresses the first, second and third polypeptides expands at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that expresses only the first polypeptide, only the second polypeptide, only the third polypeptide, the first and second polypeptide, the first and third polypeptide, the second and third polypeptide or none of the first, second and third polypeptides.

[0014] In some embodiments, the composition further comprises a fourth sequence encoding a fourth polypeptide comprising a fourth portion of the split receptor switch polypeptide. In some embodiments, the fourth polypeptide comprises: a second extracellular domain from the third cytokine receptor; and a fourth transmembrane domain. In some embodiments, the recombinant polynucleic acid further comprises a fourth sequence encoding a fourth polypeptide comprising a fourth portion of the split receptor switch polypeptide. In some embodiments, the fourth polypeptide comprises: a fourth transmembrane domain, and a second intracellular domain comprising an intracellular domain from the third cytokine receptor. In some embodiments, the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide in a cell that expresses the first, second, third and fourth polypeptide compared to a cell that expresses the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the first and second polypeptide, the first and third polypeptide, the first and fourth polypeptide, the second and third polypeptide, the second and fourth polypeptide, the third and fourth polypeptide, the first and the second and the third polypeptide, the first and the second and the fourth polypeptide, the first and the third and the fourth polypeptide, the second and the third polypeptide and the fourth polypeptide, or none of the first, second, third and fourth polypeptides. In some embodiments, the cell expressing the first, second, third and fourth polypeptides expands at least 2- fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that expresses the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the first and second polypeptide, the first and third polypeptide, the first and fourth polypeptide, the second and third polypeptide, the second and fourth polypeptide, the third and fourth polypeptide, the first and the second and the third polypeptide, the first and the second and the fourth polypeptide, the first and the third and the fourth polypeptide, the second and the third polypeptide and the fourth polypeptide, or none of the first, second, third and fourth polypeptides.

[0015] In some embodiments, a cell that does not express one or more or each of the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide does not expand in the presence of the cytokine that binds to the extracellular domain of the cytokine receptor switch polypeptide. In some embodiments, the cytokine is IL21. In some embodiments, (a) a cell that expresses the first and second polypeptide expands in the presence of IL2 and a cell that does not express the first polypeptide, the second polypeptide, or the first and second polypeptide expands in the presence of IL2; (b) a cell that expresses the first and second and third polypeptide expands in the presence of IL2 and a cell that does not express the first polypeptide, the second polypeptide, the third polypeptide, the first and second polypeptide, the first and third polypeptide, the second and third polypeptide or the first and the second and the third polypeptide expands in the presence of IL2; or (c) a cell that expresses the first and second and third and fourth polypeptide expands in the presence of IL2 and a cell that does not express the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the first and second polypeptide, the first and third polypeptide, the first and fourth polypeptide, the second and third polypeptide, the second and fourth polypeptide, the third and fourth polypeptide, the first and the second and the third polypeptide, the first and the second and the fourth polypeptide, the first and the third and the fourth polypeptide, the second and the third polypeptide and the fourth polypeptide, or the first and the second and the third polypeptide and the fourth polypeptide expands in the presence of IL2.

[0016] In some embodiments, the first transmembrane domain, the second transmembrane domain, the third transmembrane domain, and/or the fourth transmembrane domain is not from IL21R. In some embodiments, the first transmembrane domain, the second transmembrane domain, the third transmembrane domain, and/or the fourth transmembrane domain is from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, R0R1, LNGFR, IL2RG, or PDGFRa.

[0017] In some embodiments, the first extracellular domain is from IL21R and the second extracellular domain is from IL-2RG (CD 132). In some embodiments, the first intracellular domain comprises an intracellular signaling domain from IL2RB, IL7R, or IL2RG. In some embodiments, the extracellular domain of the cytokine receptor switch polypeptide comprises a sequence with at least about 80% sequence identity to any one of SEQ ID NOs: 54-61. In some embodiments, the transmembrane domain of the cytokine receptor switch polypeptide comprises a sequence with at least about 80% sequence identity to any one of SEQ ID NOs: 62-84. In some embodiments, the intracellular domain of the cytokine receptor switch polypeptide comprises a sequence with at least about 80% sequence identity to any one of SEQ ID NOs: 85-88. In some embodiments, the cytokine receptor switch polypeptide comprises a sequence with at least about 80% sequence identity to any one of SEQ ID NOs: 1-53.

[0018] Also disclosed herein is a composition comprising a recombinant polynucleic acid comprising a first sequence encoding a first polypeptide comprising a first portion of a split receptor switch polypeptide and a second sequence encoding a second polypeptide comprising a second portion of the split receptor switch polypeptide, wherein the first polypeptide comprises: (a) a first extracellular domain comprising a cytokine binding domain from a first cytokine receptor, and (b) a first transmembrane domain, wherein the second polypeptide comprises: (a) a second transmembrane domain, and (b) a first intracellular domain comprising an intracellular signaling domain from the first cytokine receptor. In some embodiments, the first cytokine receptor is an IL-2RG (CD 132) receptor. In some embodiments, the composition comprising the split receptor switch polypeptide further comprises a chimeric antigen receptor (CAR).

[0019] In some embodiments, the first protein of interest is a first CAR, the second protein of interest is a second CAR, and the third protein of interest is a third CAR. In some embodiments, the recombinant polynucleic acid comprises, in an order from 5’ end to 3’ end, the sequence encoding the CAR and the sequence encoding the first or second polypeptide. In some embodiments, the CAR comprises (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain. In some embodiments, the antigen binding domain is an anti-CD19 binding domain. In some embodiments, the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID NO: 106 or 107. In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID Nos: 120-143.

[0020] In some embodiments, the antigen binding domain of the first CAR is an anti-CD22 binding domain.

[0021] In some embodiments, the antigen binding domain of the second CAR is an anti-CD19 or anti- CD20 binding domain. In some embodiments, the antigen binding domain of the second CAR is an anti-anti-CD20 binding domain. In some embodiments, the antigen binding domain of the second CAR is an anti-CD19 binding domain and the antigen binding domain of the third CAR is an anti- CD20 binding domain.

[0022] In some embodiments, the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID NO: 108.

[0023] In some embodiments, the antigen binding domain is an anti-CD20 binding domain. In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID Nos: 144-180. In some embodiments, the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7-H3, GPC2, LI CAM, EGFR, mesothelin, MART-1, gplOO (Pmel 17), tyrosinase, TRP- 1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MO VI 8, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD 19, CD20, CD22, ROR1, CD2 and GD2. In some embodiments, the intracellular domain of the CAR comprises an intracellular signaling domain from CD2. In some embodiments, the intracellular domain of the CAR comprises an intracellular signaling domain from CD3zeta, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70. In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28. In some embodiments, the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28.

[0024] In some embodiments, the CAR comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 109-111. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a population of cells. In some embodiments, the population of cells comprises at least lxl0 ^A 5 cells. In some embodiments, at least 60% or more of the cells in the population of cells express two CARs. In some embodiments, at least 60% or more of the cells in the population of cells express three CARs. In some embodiments, composition comprises a pharmaceutical composition comprising the split receptor switch polypeptide and a pharmaceutically acceptable excipient or carrier.

[0025] Also disclosed herein is a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the method further comprises administering to the subject the cytokine that binds to the cytokine binding domain from a first cytokine receptor. In some embodiments, the method further comprises administering to the subject a cytokine that binds to the extracellular domain of the cytokine receptor switch polypeptide. In some embodiments, the method further comprises administering to the subject IL21.

[0026] In some embodiments, the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and/or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LU AD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC).

[0027] Also disclosed herein is a method of expanding cells, the method comprising culturing a cell expressing the split receptor switch polypeptide in the presence of the cytokine that binds to the extracellular domain of the cytokine receptor switch polypeptide, thereby expanding the cell. In some embodiments, the cell is expanded at least 3-fold.

[0028] Also disclosed herein is a method of making a population of chimeric antigen receptor (CAR)- expressing immune effector cells, comprising (a) contacting a cell expressing a split receptor switch polypeptide with a cytokine that binds to the cytokine binding domain from the first cytokine receptor; and (b) expanding the cell in the presence of the cytokine that binds to the cytokine binding domain from the first cytokine receptor, thereby making a population of chimeric antigen receptor (CAR)- expressing immune effector cells.

[0029] Also disclosed herein is a method of modulating activity of a chimeric antigen receptor (CAR)- expressing immune effector cell, comprising contacting a cell expressing a split receptor switch polypeptide with a cytokine that binds to the cytokine binding domain from the first cytokine receptor, wherein the cell comprises a CAR, thereby modulating activity of a chimeric antigen receptor (CAR)- expressing immune effector cell. In some embodiments, activity of a cell not expressing a CAR is modulated to a lesser extent by contacting the cell with a cytokine that binds to the cytokine binding domain from the first cytokine receptor than the extent of modulation of activity of a cell that comprises the CAR that is contacted with the cytokine.

INCORPORATION BY REFERENCE

[0030] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0032] FIG. 1 depicts exemplary structures of the split receptor switch polypeptides with pairing transmembrane domains that could be used in conditional multi-component cytokine drive to select multi-edited T cells.

[0033] FIG. 2 depicts an exemplary complex with endogenous Fey chain that is formed using the exemplary split receptor switch polypeptides depicted.

[0034] FIG. 3A shows cell expansion data of cells 14 days following transduction with the indicated split receptor switch polypeptide constructs and stimulation with IL-2 or IL-21.

[0035] FIG. 3B shows cells that stained as double positive for the indicated split receptor switch polypeptide constructs 14 days following transduction and stimulation with IL-2 or IL-21.

[0036] FIG. 4 depicts exemplary flow cytometry data of cells 14 days following transduction with the indicated split receptor switch polypeptide constructs and stimulation with IL-2 or IL-21. T cells with split IL-21/IL-2RB receptor grown with IL-21 are highly pure in double positive cells. 82% yield of double positive cells with IL-21 vs IL-2 on day 14 was observed.

[0037] FIG. 5A depicts an exemplary 2 component split receptor switch polypeptide system with swapped cytodomains.

[0038] FIG. 5B depicts an exemplary 3 component split receptor switch polypeptide system with swapped cytodomains.

[0039] FIG. 5C depicts an exemplary 4 component split receptor switch polypeptide system with swapped cytodomains. [0040] FIG. 6 shows cell expansion data of cells 14 days following transduction with the indicated cytodomain-swapped split receptor switch polypeptide constructs and stimulation with IL-2 or IL-21. [0041] FIG. 7 illustrates flow cytometry data demonstrating the expression of CD 19, CD20, and CD22 CARs in T cells transduced with a split receptor switch polypeptide comprising — a vector containing IL-21R-TREM1, a CD 19 CAR and a CD20 CAR as well as a vector containing DAP12-IL2RB and a CD22 CAR. The expression of the three CARs was measured following T cell culture grown in the presence of no cytokine (25.41% tri-CAR expression), IL-21 (65.61% tri -CAR expression), and IL-2 (21.57% tri-CAR expression).

DETAILED DESCRIPTION OF THE DISCLOSURE

[0042] Disclosed herein include compositions and methods for selective expansion of a cell population, such as a CAR-T cell population. Specifically, the present disclosure describes a split receptor switch polypeptide comprising a recombinant polynucleic acid comprising a first sequence encoding a first polypeptide comprising a first portion of a cytokine switch receptor and a second sequence encoding a second polypeptide comprising a second portion of the cytokine switch receptor, wherein the first polypeptide comprises: (a) a first extracellular domain comprising a cytokine binding domain from a first cytokine receptor, and (b) a first transmembrane domain, wherein the second polypeptide comprises: (a) a second transmembrane domain, and (b) a first intracellular domain comprising an intracellular signaling domain from a second cytokine receptor; wherein the first cytokine receptor and the second cytokine receptor are different cytokine receptors. In some embodiments, the split receptor switch polypeptide may be expressed in a cell. In some embodiments the cell expressing the split receptor switch polypeptide may further comprise a CAR.

Definitions

[0043] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”

[0044] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. [0045] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0046] “Percent (%) sequence identity” or “homology” with respect to the nucleic acid or amino acid sequences identified herein is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. [0047] “Percent (%) identity” with respect to the nucleic acid or amino acid sequences identified herein is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.

[0048] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the recombinant polypeptides, methods and other aspects belong. Although any recombinant polypeptides, methods and other aspects similar or equivalent to those described herein can also be used in the practice or testing of the recombinant polypeptides, methods and other aspects, representative illustrative recombinant polypeptides, methods and other aspects are now described.

[0050] The term “binding domain”, as used herein, refers to a protein, or polypeptide sequence, which specifically binds to a target.

[0051] As used herein, the term “CAR-T cells” means a T cell or population thereof, which has been modified through molecular biological methods to express a chimeric antigen receptor (CAR) on the T cell surface. The CAR is a recombinant polypeptide having a pre-defined binding specificity to a desired target which is operably connected to the intracellular part of a T-cell activation domain. By bypassing MHC-class I and class II restriction, CAR engineered T cells of both CD8+ and CD4+ subsets can be recruited for redirected target cell recognition. For examples, some CARs are fusions of immuoglobulin binding functionality (e.g., as a single-chain variable fragment (scFv) derived from a monoclonal antibody) to CD3-zeta (CD3Q transmembrane and endodomain. Such molecules result in the transmission of a zeta signal in response to recognition by the immuoglobulin binding functionality of its target.

[0052] As used herein, a subject is “in need of’ a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

[0053] As used herein, the term “operably connected” or “operably linked” refers to positioning of components such that they function in their intended manner. For example, the components can be operably connected by a fusion, a linker, and/or a spacer.

[0054] As used herein, “specifically binds” means that the binding domain preferentially binds the corresponding target over other proteins. In some embodiments, “specifically binds” means that the binding domains have a higher affinity for the target than for other proteins. In some embodiments, the binding domain is a cytokine binding domain and the target is the corresponding cytokine.

[0055] As used herein, a “therapeutically effective amount” or “therapeutically effective number” of an agent is an amount or number sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of an agent means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.

[0056] As used herein, the term “treat,” “treating” or “treatment” of any disease or disorder refers, in one instance, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another instance, “treat”, “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another instance, “treat”, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.

[0057] It is appreciated that certain features of the recombinant polypeptides, and/or recombinant nucleic acids encoding the recombinant polypeptides, methods and other aspects, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the recombinant polypeptides, and/or recombinant nucleic acids encoding the recombinant polypeptides, methods and other aspects, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present recombinant polypeptides, methods and other aspects and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

Split receptor switch polypeptides.

[0058] In some aspects, the present disclosure describes a composition comprising a split receptor switch polypeptide comprising a recombinant polynucleic acid comprising a first, second, third, and/or fourth sequence encoding a first, second, third, and/or fourth polypeptide, wherein the composition may further comprise recombinant polynucleic acid comprising a first, second, third, and/or fourth chimeric antigen receptor (CAR), wherein the first, second, third, and/or fourth polypeptide may comprise the CAR. The first, second, third, and/or fourth polypeptides may comprise an extracellular domain and/or a CAR, an extracellular domain and a transmembrane domain and/or a CAR, an extracellular domain and a transmembrane domain and an intracellular domain and/or a CAR, a transmembrane domain and/or a CAR, a transmembrane domain and an intracellular domain and/or a CAR, or an intracellular domain and/or a CAR.

Extracellular Domain

[0059] The extracellular domain of the split receptor switch polypeptide functions to receive the cytokine signal outside of a cell by specifically binding to the cytokine. In some embodiments, the extracellular domain of the split receptor switch polypeptide may comprise the extracellular domain of the first polypeptide. In some embodiments, the extracellular domain of the split receptor switch polypeptide may comprise the extracellular domain of the second polypeptide. In some embodiments, the extracellular domain of the split receptor switch polypeptide may comprise the extracellular domain of the third polypeptide. In some embodiments, the extracellular domain of the split receptor switch polypeptide may comprise the extracellular domain of the fourth polypeptide. In some embodiments, the extracellular domain of the split receptor switch polypeptide may comprise the extracellular domain of the first, second, third, and/or fourth polypeptide, and/or any combination thereof. In some embodiments, the extracellular domain comprises a cytokine binding domain from a cytokine receptor. In some embodiments, the cytokine receptor is an interleukin 21 (IL21) receptor. In some embodiments, the cytokine receptor is an IL-2RG (CD 132) receptor. In some embodiments, the cytokine receptor is IL2Ra, IL2Rb, IL4R, IL7R, IL9R, and/or IL15R. In some embodiments, the cytokine binding domain from the cytokine receptor can specifically bind to a cytokine. In some embodiments, the cytokine binding domain from the cytokine receptor specifically binds to IL21. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 50% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 60% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 70% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 80% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 54. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 54.

[0060] The cytokine binding domain from the cytokine receptor can specifically bind to the cytokine. In some embodiments, the cytokine binding domain from the cytokine receptor specifically binds to IL21 and/or IL2. In some embodiments, the cytokine binding domain from the cytokine receptor is an IL2RG extracellular domain. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 50% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 60% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 70% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 80% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 55. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 55.

[0061] The cytokine binding domain from the cytokine receptor can specifically bind to the cytokine. In some embodiments, the cytokine binding domain from the cytokine receptor specifically binds to IL21 and/or IL2. In some embodiments, the cytokine binding domain is IL2Ra. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 50% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 60% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least X0% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 80% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 9X% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 56. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 56. [0062] The cytokine binding domain from the cytokine receptor can specifically bind to the cytokine. In some embodiments, the cytokine binding domain from the cytokine receptor specifically binds to IL21 and/or IL2. In some embodiments, the cytokine receptor is IL2Rb. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 50% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 60% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 70% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 80% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 57. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 57.

[0063] The cytokine binding domain from the cytokine receptor can specifically bind to the cytokine. In some embodiments, the cytokine binding domain from the cytokine receptor specifically binds to IL4. In some embodiments the cytokine binding domain is IL4R. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 50% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 60% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 70% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 80% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 58. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 58.

[0064] The cytokine binding domain from the cytokine receptor can specifically bind to the cytokine. In some embodiments, the cytokine binding domain from the cytokine receptor specifically binds to IL7. In some embodiments, the cytokine binding domain is IL7R. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 50% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 60% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 70% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 80% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 59. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 59.

[0065] The cytokine binding domain from the cytokine receptor can specifically bind to the cytokine. In some embodiments, the cytokine binding domain from the cytokine receptor specifically binds to IL9. In some embodiments, the cytokine receptor is IL9R. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 50% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 60% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 70% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 80% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 60. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 60.

[0066] The cytokine binding domain from the cytokine receptor can specifically bind to the cytokine. In some embodiments, the cytokine binding domain from the cytokine receptor specifically binds to IL15. In some embodiments, the cytokine binding domain is IL15R. In some embodiments, the cytokine receptor is IL9R. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 50% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 60% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 70% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists an amino acid sequence with at least 80% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 61. In some embodiments, the cytokine binding domain from the cytokine receptor consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 61.

[0067] In some embodiments, the extracellular domain of the split receptor switch polypeptide may comprise one, two , three, and/or four extracellular domains. In some embodiments, one, two , three, and/or four extracellular domains may comprise the extracellular domain of the first, second, third, and/or fourth polypeptides. In some embodiments, any one of SEQ ID NOs: 54-61 may comprise the extracellular domain of the first polypeptide. In some embodiments, any one of SEQ ID NOs: 54-61 may comprise the extracellular domain of the second polypeptide. In some embodiments, any one of SEQ ID NOs: 54-61 may comprise the extracellular domain of the third polypeptide. In some embodiments, any one of SEQ ID NOs: 54-61- may comprise the extracellular domain of the fourth polypeptide.

Transmembrane Domain

[0068] In some embodiments, the extracellular domain of the split receptor switch polypeptide is operably connected to a transmembrane domain. In some embodiments, the extracellular domain is connected to the transmembrane domain by a spacer. In some embodiments, the transmembrane domain of the split receptor switch polypeptide serves to transduce the cytokine signal received by the extracellular domain to the intracellular domain. In some embodiments, the transmembrane domain may lack an intracellular domain. In some embodiments, the transmembrane domain of the split receptor switch polypeptide may comprise the transmembrane domain of the first polypeptide. In some embodiments, the transmembrane domain of the split receptor switch polypeptide may comprise the transmembrane domain of the second polypeptide. In some embodiments, the transmembrane domain of the split receptor switch polypeptide may comprise the transmembrane domain of the third polypeptide. In some embodiments, the transmembrane domain of the split receptor switch polypeptide may comprise the transmembrane domain of the fourth polypeptide. In some embodiments, the transmembrane domain may form a multimer. The transmembrane domain can be any suitable transmembrane domain known in the art. The transmembrane domain can be selected from a transmembrane region of a transmembrane protein such as, for example, Type I transmembrane proteins, an artificial hydrophobic sequence or a combination thereof. Examples of the transmembrane domain include may include the transmembrane regions of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Synthetic transmembrane domains may comprise a triplet of phenylalanine, tryptophan and valine. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the intracellular signaling domain of the split receptor switch polypeptide. In some embodiments, a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain. In some embodiments, the transmembrane domain is an IL21 receptor transmembrane domain. In some embodiments, the transmembrane domain is an IL7 receptor transmembrane domain. In some embodiments, the transmembrane domain is an IL2 receptor beta transmembrane domain. In some embodiments, the transmembrane domain is an UG2B17 transmembrane domain. In some embodiments, the transmembrane domain is an Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) transmembrane domain. In some embodiments, the transmembrane domain is an DNAX Activating Protein of 12 kDA (DAP12) transmembrane domain. In some embodiments, the transmembrane domain is an IL2RG, IL2Ra, and/ or IL2Rb transmembrane domain. In some embodiments, the transmembrane domain is an CD4 transmembrane domain. In some embodiments, the transmembrane domain is an CD4 transmembrane domain. In some embodiments, the transmembrane domain is an CD8a transmembrane domain. In some embodiments, the transmembrane domain is an CD8P transmembrane domain. In some embodiments, the transmembrane domain is an CD38 transmembrane domain. In some embodiments, the transmembrane domain is an CD3s transmembrane domain. In some embodiments, the transmembrane domain is a Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1) transmembrane domain. In some embodiments, the transmembrane domain is a Low Affinity Nerve Growth Factor Receptor (LNGFR) transmembrane domain. In some embodiments, the transmembrane domain is a platelet derived growth factor receptor alpha (PDGFRa) transmembrane domain. In some embodiments, the transmembrane domain is an EPOR transmembrane domain. In some embodiments, the transmembrane domain is a glycophorin A transmembrane domain. In some embodiments, the transmembrane domain is a DAP 10 transmembrane domain. In some embodiments, the transmembrane domain is an FGFR1 transmembrane domain. In some embodiments, the transmembrane domain is a KIR2DS2 transmembrane domain.

[0069] In some embodiments, the transmembrane domain comprises the transmembrane domain of IL21R. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 62. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 62. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 62. In some embodiments, the transmembrane domain comprises the transmembrane domain of IL7.

[0070] In some embodiments, the transmembrane domain comprises a transmembrane domain from IL7R. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 63. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 63. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 63. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

- l- 63. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 63.

[0071] In some embodiments, the transmembrane domain comprises the transmembrane domain of IL2RB. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 64. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 64. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 64. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

64. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 64.

[0072] In some embodiments, the transmembrane domain comprises the transmembrane domain of UG2B17. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 65. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 65. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 65. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

65. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 65.

[0073] In some embodiments, the transmembrane domain comprises the transmembrane domain of TREM1 . In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 66. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 66. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 66. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

66. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 66.

[0074] In some embodiments, the transmembrane domain comprises the transmembrane domain of DAP12. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 67. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 67. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 67. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

67. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 67.

[0075] In some embodiments, the transmembrane domain comprises the transmembrane domain of IL2RG. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 68. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 68. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 68. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

68. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 68. [0076] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD4. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 69. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 69. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 69. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 69.

[0077] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8a. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 70. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 70. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 70. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

70. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 70.

[0078] In some embodiments, the transmembrane domain comprises the transmembrane domain of EPOR. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 71. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 71. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 71. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

71. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 71.

[0079] In some embodiments, the transmembrane domain comprises the transmembrane domain of Glycophorin A. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 72. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 72. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 72. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

72. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 72.

[0080] In some embodiments, the transmembrane domain comprises the transmembrane domain of IL2Ra. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 73. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 73. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 73. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

73. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 73.

[0081] In some embodiments, the transmembrane domain comprises the transmembrane domain of IL2RB. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 74. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 74. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 74. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 74.

[0082] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 75. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 75. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 75. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

75. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 75.

[0083] In some embodiments, the transmembrane domain comprises the transmembrane domain of DAP 10. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 76. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 76. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 76. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

76. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 76.

[0084] In some embodiments, the transmembrane domain comprises the transmembrane domain of FGFR1. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 77. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 77. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 77. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

77. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 77.

[0085] In some embodiments, the transmembrane domain comprises the transmembrane domain of KIR2DS2. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 78. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 78. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 78. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

78. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 78.

[0086] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8B. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 79. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 79. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 79. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 79.

[0087] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD33. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 80. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 80. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 80.

[0088] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD3E. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 81. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 81. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

81. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 81.

[0089] In some embodiments, the transmembrane domain comprises the transmembrane domain of ROR1. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 82. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 82. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 82. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

82. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 82.

[0090] In some embodiments, the transmembrane domain comprises the transmembrane domain of LNGFR. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 83. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 83. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 83. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO:

83. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 83.

[0091] In some embodiments, the transmembrane domain comprises the transmembrane domain of PDGFRa. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 84. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 50% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 60% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 70% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain consists an amino acid sequence with at least 80% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain consists of an amino acid sequence of SEQ ID NO: 84. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 84. In some embodiments, the transmembrane domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 84.

[0092] In some embodiments, the transmembrane domain of the split receptor switch polypeptide may comprise one, two , three, and/or four transmembrane domains. In some embodiments, one, two , three, and/or four transmembrane domains may comprise the transmembrane domain of the first, second, third, and/or fourth polypeptides. In some embodiments, any one of SEQ ID NOs: 62-84 may comprise the transmembrane domain of the first polypeptide. In some embodiments, any one of SEQ ID NOs: 62-84 may comprise the transmembrane domain of the second polypeptide. In some embodiments, any one of SEQ ID NOs: 62-84 may comprise the transmembrane domain of the third polypeptide. In some embodiments, any one of SEQ ID NOs: 62-84 may comprise the transmembrane domain of the fourth polypeptide. In some embodiments, the first transmembrane domain interacts with the second transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain form a multimer. In some embodiments, the first transmembrane domain and the second transmembrane domain form a homodimer or a heterodimer. In some embodiments, the third transmembrane domain interacts with the first transmembrane domain and/or the second transmembrane domain. In some embodiments, the first transmembrane domain, the second transmembrane domain and the third transmembrane domain form a multimer. In some embodiments, the third transmembrane domain interacts with the first transmembrane domain but not the second transmembrane domain. In some embodiments, the first transmembrane domain and the third transmembrane domain form a multimer. In some embodiments, the third transmembrane domain interacts with the second transmembrane domain but not the first transmembrane domain. In some embodiments, the second transmembrane domain and the third transmembrane domain form a multimer. In some embodiments, the first transmembrane domain, the second transmembrane domain, the third transmembrane domain, and/or the fourth transmembrane domain is not from IL21R. In some embodiments, the first transmembrane domain, the second transmembrane domain, the third transmembrane domain, and/or the fourth transmembrane domain is from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, R0R1, LNGFR, IL2RG, or PDGFRa.

Intracellular Domain

[0093] In some embodiments, the split receptor switch polypeptide comprises an intracellular domain. In some embodiments, the intracellular domain serves to transduce the received cytokine signal to activate the downstream signaling cascade. In some embodiments, the intracellular domain comprises a first, second, and/or third intracellular signaling domain, from a first, second, and/or third cytokine receptor. In some embodiments, the intracellular domain comprises an intracellular signaling domain from a second cytokine receptor, wherein the first cytokine receptor and the second cytokine receptor are different cytokine receptors. In some embodiments, the intracellular domain comprises an intracellular signaling domain from the first cytokine receptor of the extracellular domain. In some embodiments, the first intracellular signaling domain is activated in the presence of a cytokine that binds to the extracellular domain of the first polypeptide. In some embodiments, wherein the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that does not express the first polypeptide, the second polypeptide or both in the presence of the cytokine. In some embodiments, the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that does not express a IL-2RG (CD 132) receptor. In some embodiments, the intracellular domain comprises a second intracellular domain comprising an intracellular domain from a third cytokine receptor. In some embodiments, the second intracellular domain comprises an intracellular domain from the first cytokine receptor extracellular domain. In some embodiments, the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that does not express the first polypeptide, the second polypeptide, the third polypeptide, the first and second polypeptide, the first and third polypeptide, the second and third polypeptide or the first and the second and the third polypeptide in the presence of the cytokine. In some embodiments, the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that does not express the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the first and second polypeptide, the first and third polypeptide, the first and fourth polypeptide, the second and third polypeptide, the second and fourth polypeptide, the third and fourth polypeptide, the first and the second and the third polypeptide, the first and the second and the fourth polypeptide, the first and the third and the fourth polypeptide, the second and the third polypeptide and the fourth polypeptide, or the first and the second and the third polypeptide and the fourth polypeptide in the presence of the cytokine. In some embodiments, the first cytokine receptor is an IL21 receptor and the second cytokine receptor is a cytokine receptor other than an IL21 receptor. In some embodiments, the intracellular signaling domain from the second cytokine receptor induces a downstream signaling that enhances cytotoxicity, promotes stem memory T cells (T _SC m) formation, and/or promotes central memory T cells (T _cm ) formation. In some embodiments, the intracellular signaling domain from the second cytokine receptor induces STAT5 signaling. In some embodiments, the second cytokine receptor is IL2 receptor beta (IL2RB). In some embodiments, the second cytokine receptor IL7 receptor alpha (IL7Ra). In some embodiments, the second cytokine receptor is IL7 receptor. In some embodiments, the second cytokine receptor is IL9 receptor. In some embodiments the second cytokine receptor is IL2RG (CD 132) receptor. In some embodiments the second cytokine receptor is IL21 receptor. In some embodiments the second cytokine receptor is a IL2 receptor. In some embodiments, the second cytokine receptor is an IL9 receptor. In some embodiments, the intracellular signaling domain comprises an IL7R. In some embodiments, the intracellular signaling domain comprises an IL7Ra. In some embodiments, the intracellular signaling domain comprises an IL2RB. In some embodiments, the intracellular signaling domain comprises an IL9R. In some embodiments, the intracellular signaling domain comprises an IL2RG. In some embodiments, the intracellular signaling domain comprises an IL21R. In some embodiments, the intracellular signaling domain comprises an IL2R.

[0094] In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of IL7R. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 50% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 60% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 70% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 80% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 96% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 97% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 98% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 99% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 99.5% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 96% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 97% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 98% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 99% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 99.5% identity of SEQ ID NO: 85. In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of IL2RB. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 50% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 60% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 70% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 80% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 96% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 97% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 98% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 99% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 99.5% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 96% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 97% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 98% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 99% identity of SEQ ID NO: 86. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 99.5% identity of SEQ ID NO: 86.

[0095] In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of IL9R. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 50% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 60% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 70% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 80% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 96% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 97% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 98% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 99% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 99.5% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 96% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 97% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 98% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 99% identity of SEQ ID NO: 87. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 99.5% identity of SEQ ID NO: 87.

[0096] In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of IL2RG. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 50% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 60% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 70% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with at least 80% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 50% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 60% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 70% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with at least 80% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 90% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 95% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 96% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 97% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 98% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 99% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain comprises an amino acid sequence with least 99.5% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 90% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 95% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 96% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 97% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 98% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 99% identity of SEQ ID NO: 88. In some embodiments, the intracellular signaling domain consists of an amino acid sequence with least 99.5% identity of SEQ ID NO: 88.

Protease Cleavage Sites

[0097] In some embodiments, a protease cleavage site is disposed between the protein localization tag and the split receptor switch polypeptide. In some embodiments, the protease cleavage site is disposed between the protein localization tag and the intracellular domain of the split receptor switch polypeptide. In some embodiments, the protease cleavage site is disposed between the protein localization tag and the extracellular domain of the split receptor switch polypeptide. Protease cleavage sites are to be understood as amino acid residues that are recognized by proteases and/or amino acid residues whose peptide bond is cleaved by proteases. In some embodiments, a protease cleavage site can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. Optionally, additional amino acids can be present at the N-terminus and/or C-terminus of the cleavage site. A protease cleavage site also can be a variant of a cleavage site of a known protease as long as it is recognized/cleaved by the protease.

[0098] Various protease cleavage sites include, but are not limited to protease cleavage sites for proteases from the serine protease family, or for metalloproteases, or for a protease from the cysteine protease family, and/or the aspartic acid protease family, and/or the glutamic acid protease family. In some embodiments, serine proteases cleavage sites include, but are not limited to, cleavage sites for chymotrypsin-like proteases, and/or subtili sin-like proteases, and/or alpha/beta hydrolases, and/or signal peptidases. In some embodiments, metalloprotease recognition sites include, but are not limited to, cleavage sites for metallocarboxypeptidases or metalloendopeptidases. In some embodiments, the protease cleavage site is TEV protease cleavage site.

Embodiments

Two Component

[0099] In some embodiments, the split receptor switch polypeptide may be a two-component split receptor switch polypeptide. In some embodiments, the two component split receptor switch polypeptide may comprise a first polypeptide comprising a first extracellular domain comprising a cytokine binding domain from IL21R, IL2RG, IL2R, IL4R, IL7R, IL9R or IL15R, and a first transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a second polypeptide comprising a second transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a first intracellular domain comprising an intracellular signaling domain from a second cytokine receptor, wherein the intracellular signaling domain is an IL7R, IL7Ra, IL2RB, IL9R, IL2RG, IL21R, or IL2R intracellular signaling domain. In some embodiments the first and second cytokine receptors are different cytokine receptors. In some embodiments, the first and second cytokine receptors are the same cytokine receptors. In some embodiments the first polypeptide and the second polypeptide form a complex comprising the cytokine switch receptor. In some embodiments, the first polypeptide and the second polypeptide form a complex. In some embodiments, the cytokine switch receptor is a functional cytokine switch receptor. In some embodiments, the first transmembrane domain interacts with the second transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain form a multimer. In some embodiments, the first transmembrane domain and the second transmembrane domain form a homodimer or a heterodimer. In some embodiments, the second polypeptide is not a functional cytokine switch receptor. In some embodiments the cytokine switch receptor is a functional cytokine switch receptor capable of signaling to the cell. In some embodiments the first polypeptide is not a functional cytokine switch receptor capable of signaling to the cell. In some embodiments, the second polypeptide is not a functional cytokine switch receptor capable of signaling to the cell. In some embodiments, the first intracellular signaling domain is activated in the presence of a cytokine that binds to the extracellular domain of the first polypeptide. In some embodiments the two-component split receptor switch polypeptide may be expressed in a cell. In some embodiments, the cell expresses endogenous IL2RG. In some embodiments, the endogenous IL2RG receptor forms a complex with the first polypeptide, the second polypeptide or both. In some embodiments, the first polypeptide further comprises an intracellular domain from a third cytokine receptor. In some embodiments, the intracellular domain from the third cytokine receptor may comprise an intracellular signaling domain from IL7R, IL7Ra, IL2RB, IL9R, IL2RG, IL21R, or IL2R. In some embodiments, the intracellular signaling domain from the third cytokine receptor is different than the first cytokine receptor and the second cytokine receptor. In some embodiments, the second polypeptide further comprises a second extracellular domain from a third cytokine receptor. In some embodiments, the second extracellular domain from a third cytokine receptor comprises a cytokine binding domain from IL21R, IL2RG, IL2R, IL4R, IL7R, IL9R or IL15R. In some embodiments, the second extracellular domain from a third cytokine receptor comprises a cytokine receptor that is different than the first cytokine receptor and the second cytokine receptor. In some embodiments, the first extracellular domain of the first polypeptide comprises a first portion of the cytokine binding domain, and wherein the second extracellular domain of the third polypeptide comprises a second portion of the cytokine binding domain, wherein the cytokine binding domain comprises a IL21R, IL2RG, IL2R, IL4R, IL7R, IL9R or IL15R.

Three Component

[0100] In some embodiments, the split receptor switch polypeptide may be a three-component split receptor switch polypeptide. In some embodiments, the three-component split receptor switch polypeptide may comprise a first polypeptide comprising a first extracellular domain comprising a cytokine binding domain from IL21R, IL2RG, IL2R, IL4R, IL7R, IL9R or IL15R, and a first transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a second polypeptide comprising a second transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a first intracellular domain comprising an intracellular signaling domain from a second cytokine receptor, wherein the intracellular signaling domain is an IL7R, IL7Ra, IL2RB, IL9R, IL2RG, IL21R, or IL2R intracellular signaling domain and a third polypeptide comprising a third transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, R0R1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a second intracellular domain comprising an intracellular domain from a third cytokine receptor, wherein the intracellular signaling domain is an IL7R, IL7Ra, IL2RB, IL9R, IL2RG, IL21R, or IL2R intracellular signaling domain. In some embodiments, the third cytokine receptor of the three-component split receptor switch polypeptide is different than the first cytokine receptor and/or the second cytokine receptor. In some embodiment, the third polypeptide of the three-component split receptor polypeptide comprises a second extracellular domain from a third cytokine receptor, wherein the extracellular domain is a cytokine binding domain from IL21R, IL2RG, IL2R, IL4R, IL7R, IL9R or IL15R, and a third transmembrane domain, wherein the third transmembrane domain comprises a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, R0R1, LNGFR, IL2RG, IL7R, IL2RB, UG2B 17, or PDGFRa. In some embodiments the third cytokine binding domain of the second extracellular domain is different than the first or second cytokine receptor. In some embodiments, the third transmembrane domain of the three-component split receptor switch polypeptide interacts with the first transmembrane domain and/or the second transmembrane domain. In some embodiments, the third transmembrane domain interacts with the first transmembrane domain but not the second transmembrane domain. In some embodiments, wherein the first transmembrane domain and the third transmembrane domain form a multimer. In some embodiments, the third transmembrane domain interacts with the second transmembrane domain but not the first transmembrane domain. In some embodiments, the second transmembrane domain and the third transmembrane domain form a multimer.

Four Component

[0101] In some embodiments, the split receptor switch polypeptide may be a four-component split receptor switch polypeptide. In some embodiments, the three-component split receptor switch polypeptide may comprise a first polypeptide comprising a first portion of the split receptor comprising a first extracellular domain comprising a cytokine binding domain from IL21R, IL2RG, IL2R, IL4R, IL7R, IL9R or IL15R, and a first transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a second polypeptide comprising a second portion of the split receptor switch polypeptide comprising a second transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a first intracellular domain comprising an intracellular signaling domain from a second cytokine receptor, wherein the intracellular signaling domain is an IL7R, IL7Ra, IL2RB, IL9R, IL2RG, IL21R, or IL2R intracellular signaling domain, and a third polypeptide comprising a third portion of the split receptor switch polypeptide comprising a third transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a second intracellular domain comprising an intracellular domain from a third cytokine receptor, wherein the intracellular signaling domain is an IL7R, IL7Ra, IL2RB, IL9R, IL2RG, IL21R, or IL2R intracellular signaling domain, and a fourth polypeptide comprising a fourth portion of the split reception switch polypeptide comprising a second extracellular domain from the third cytokine receptor wherein the second extracellular domain is a cytokine binding domain from IL21R, IL2RG, IL2R, IL4R, IL7R, IL9R or IL15R, and a fourth transmembrane domain, comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B 17, or PDGFRa. In some embodiments, the four-component split receptor switch polypeptide comprises the first and second polypeptides, and a third polypeptide wherein the third polypeptide comprises a second extracellular domain from a third cytokine receptor, wherein the second extracellular domain is a cytokine binding domain from IL21R, IL2RG, IL2R, IL4R, IL7R, IL9R or IL15R, and a third transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a fourth polypeptide comprising a fourth transmembrane domain comprising a transmembrane domain from CD8a, CD8b, CD4, CD28, glycophorin A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, ROR1, LNGFR, IL2RG, IL7R, IL2RB, UG2B17, or PDGFRa, and a second intracellular domain comprising an intracellular domain from the third cytokine receptor, wherein the second intracellular domain is from an IL7R, IL7Ra, IL2RB, IL9R, IL2RG, IL21R, or IL2R intracellular signaling domain. [0102] In some embodiments, the first transmembrane domain, the second transmembrane domain, the third transmembrane domain, and/or the fourth transmembrane domain of the two, three, and/or four-component split receptor switch polypeptides is not from IL21R.

[0103] In some embodiments, the first, second, third, and/or fourth polypeptides of the two, three, and/or four-component split receptor switch polypeptides further comprise a gene of interest. In some embodiments, the gene of interest is a chimeric antigen receptor. In some embodiments, the split receptor switch polypeptide comprises, in an order from 5’ end to 3’ end, the sequence encoding the CAR and the sequence encoding the first and second polypeptides, and/or the first second and third, and/or the first, second, third, and fourth polypeptides.

[0104] In some embodiments, the recombinant polynucleic acid comprising the split receptor switch polypeptide comprises a first recombinant polynucleic acid molecule encoding the first polypeptide, a second recombinant polynucleic acid molecule encoding the second polypeptide, a third recombinant polynucleic acid molecule encoding the third polypeptide, and/or a fourth recombinant polynucleic acid molecule encoding the fourth polypeptide.

[0105] In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 1. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 1. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 2. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 2. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 3. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 3. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 4. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 4. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 5. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 5. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 6. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 6. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 7. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 7. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 8. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 8. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 9. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 9. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 10. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 10. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 11. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 11. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 12. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 12. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 13. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 13. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 14. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 14. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 15. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 15. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 16. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 16. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 17. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 17. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 18. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 18. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 19. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 19. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 20. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 20. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 21. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 21. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 22. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 22. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 23. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 23. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 24. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 24. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 25. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 25. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 26. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 26. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 27. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 27. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 28. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 28. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 29. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 29. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 30. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 30. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 31. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 31. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 32. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 32. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 33. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 33. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 34. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 34. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 35. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 35. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 36. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 36. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 37. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 37. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 38. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 38. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 39. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 39. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 40. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 40. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 41. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 41. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 42. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 42. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 43. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 43. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 44. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 44. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 45. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 45. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 46. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 46. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 47. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 47. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 48. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 48. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 49. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 49. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 50. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 50. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 51. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 51. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 52. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 52. In some embodiments, the split receptor switch polypeptide comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 53. In some embodiments, the split receptor switch polypeptide consists of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of SEQ ID NO: 53.

Polynucleotides

[0106] In some aspects, the present disclosure describes polynucleic acid sequences encoding any of the split receptor switch polypeptides disclosed herein.

[0107] In some embodiments, the recombinant polynucleic acid further comprises a sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the first, second, third, and/or fourth polypeptides of the split receptor switch receptor may further comprise a CAR. In some embodiments, the first polypeptide further comprises a first CAR. In some embodiments, the second polypeptide further comprises a second CAR. In some embodiments, the third polypeptide further comprises a third CAR. In some embodiments, the fourth polypeptide further comprises a fourth CAR. In some embodiments, the first polypeptide, and the first CAR are expressed as a single polypeptide molecule. In some embodiments, the first polypeptide is linked to the sequence encoding the first CAR by a sequence encoding a linker. In some embodiments, the second polypeptide, and the second CAR are expressed as a single polypeptide molecule. In some embodiments, the second polypeptide is linked to the sequence encoding the second CAR by a sequence encoding a linker. In some embodiments, the third polypeptide, and the third CAR are expressed as a single polypeptide molecule. In some embodiments, the third polypeptide is linked to the sequence encoding the third CAR by a sequence encoding a linker. In some embodiments, the fourth polypeptide, and the fourth CAR are expressed as a single polypeptide molecule. In some embodiments, the fourth polypeptide is linked to the sequence encoding the fourth CAR by a sequence encoding a linker. In some embodiments, the CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises the sequences in the following order (from 5’ end to 3’ end): the sequence encoding the CAR and the sequence encoding the split receptor switch polypeptide. The sequence encoding the CAR can be linked to the sequence encoding the split receptor switch polypeptide by a linker comprising or consisting of a sequence encoding a self-cleaving peptide. In some embodiments, the linker is a P2A cleavable linker a protease cleavage site, or an IRES.

[0108] The extracellular domain of the CAR comprises an antigen binding domain. The antigen binding domain can be any domain that specifically binds to an antigen. In some embodiments, the antigen is an antigen expressed by a tumor cell. In some embodiments, the antigen binding domain comprises a scFv, a nanobody, a ligand, or a receptor.

[0109] The antigen binding domain can be any molecule that binds to the selected antigen with sufficient affinity and specificity, and is often an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab')2 fragment, nanobody, diabody, or the like. Alternatively, the antigen binding domain can be a receptor or a receptor fragment that binds specifically to the target antigen. The antigen binding domain can be attached to the rest of the receptor directly (covalently) or indirectly (for example, through the noncovalent binding of two or more binding partners). Antibody derivatives are molecules that resemble antibodies in their mechanism of ligand binding, and include, for example, nanobodies, duobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, Fab fragments, single chain variable fragments (scFv), single domain antibodies (sdAb), and functional fragments thereof. See for example, D.L. Porter et al., N Engl J Med ( 2011) 365(8):725-33 (scFv); E.L. Smith et al, Mol Ther (2018)26(6): 1447-56 (scFv); S.R. Banihashemi et al., Iran J Basic Med Sci (2018) 21(5):455-64 (CD19 nanobody); F. Rahbarizadeh et al Adv Drug Deliv Rev (2019) 141 :41-46 (sdAb);S.M. Kipriyanov et al., Int J Cancer (1998) 77(5):763-72 (diabody); F. Le Gall et al., FEBS Lett (1999) 453(1-2): 164-68 (triabody); M.A. Ghetie et al., Blood (1994) 83(5): 1329-36 (F(ab')2); and M.A. Ghetie et al., Clin Cancer Res (1999) 5(12):3920-27 (F(ab')2 and Fab'). Antibody derivatives can also be prepared from therapeutic antibodies, for example without limitation, by preparing a nanobody, duobody, diabody, triabody, minibody, F(ab')2 fragment, Fab fragment, single chain variable fragment (scFv), or single domain antibody (sdAb) based on a therapeutic antibody. Antibody derivatives can also be designed using phage display techniques (see, e.g., E. Romao et al., Curr Pharm Des (2016) 22(43):6500-18).

[0110] In some embodiments, the antigen binding domain specifically binds to CD 19. In some embodiments, the antigen binding domain is an anti-CD19 binding domain. In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity to DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFS GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT. In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having at least about 80% sequence identity to EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYN S ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC AKHYYYGGS YAMDYWGQGTS VT VS S .

[OHl] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFS GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSE VK LQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KS RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

[0112] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSG GGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKL LI YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT.

[0113] In some embodiments, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 106. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 106. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 106.

[0114] In some embodiments, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 107. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 107. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 107.

[0115] In some embodiments, the antigen binding domain comprises an amino acid sequence of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 98% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 90% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 96% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of any one of SEQ ID NOs: 120-143. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of any one of SEQ ID NOs: 120-143.

[0116] In some embodiments, the antigen binding domain specifically binds to CD22. In some embodiments, the antigen binding domain is an anti-CD22 binding domain.

[0117] In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and OQSYSIPOT, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASOTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPS RFS GRGSGTDFTLTISSLQAEDFATYYCOQSYSIPOTFGQGTKLEI.

[0118] In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVT GDLED AFDI, respectively. In some embodiments, the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having at least about 80% sequence identity to

OVOLOQSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRS KWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVT V SS.

[0119] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVT V SSGGGGSDIOMTOSPSSLSASVGDRVTITCRASQTIWSYLNWYOQRPGKAPNLLIYAASS LO SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCOQSYSIPOTFGQGTKLEIK. [0120] In some embodiments, the antigen binding domain comprises an scFv with at least about 80% sequence identity to

DIOMTOSPSSLSASVGDRVTITCRASQTIWSYLNWYOQRPGKAPNLLIYAASSLOSG VPSRFS GRGSGTDFTLTISSLOAEDFATYYCOOSYSIPOTFGOGTKLEIKGGGGSOVOLOOSGPGL VK PSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITI NP DT SKNQF SLQLNS VTPEDT AVYYC ARE VTGDLED AFDIWGQGTM VT VS S . In some embodiments, the antigen binding domain comprises an scFv with at least about 85, 90, 95, 97, 98, or 99% sequence identity to

OVOLOQSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRS KWYN DYAVSVKSRITINPDTSKNOFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVT V SSGGGGSDIOMTOSPSSLSASVGDRVTITCRASOTIWSYLNWYOQRPGKAPNLLIYAASS LO SGVPSRFSGRGSGTDFTLTISSLOAEDFATYYCOQSYSIPOTFGOGTKLEIK. In some embodiments, the antigen binding domain comprises an scFv with 100% sequence identity OVOLOQSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRSKWY N DYAVSVKSRITINPDTSKNOFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGOGTMVT V SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASOTIWSYLNWYQQRPGKAPNLLIYAASS LQ SGVPSRFSGRGSGTDFTLTISSLOAEDFATYYCOQSYSIPOTFGOGTKLEIK.

[0121] In some embodiments, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 108. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 108.

[0122] In some embodiments, the antigen binding domain specifically binds to CD20.

[0123] In some embodiments, the antigen binding domain comprises an amino acid sequence of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 98% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 90% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 96% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of any one of SEQ ID NOs: 144-180. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of any one of SEQ ID NOs: 144-180.

[0124] In some embodiments, the antigen binding domain binds to an antigen that is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN- CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B7- H3, GPC2, LI CAM, EGFR, mesothelin, MART-1, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, CD2, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catenin, CDK4, Mum-1, pl5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MO VI 8, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD 19, CD20, CD22, ROR1, and GD2, or a mixture thereof.

[0125] In some embodiments, the antigen binding domain of a CAR provided herein is operatively linked to a transmembrane domain by a hinge domain. In some embodiments, the antigen binding domain of a CAR provided herein is directly linked to a transmembrane domain by a hinge domain. In some embodiments, the hinge domain of a CAR provided herein is from CD28. In some embodiments, the hinge domain of a CAR provided herein has the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP. In some embodiments, the hinge domain of a CAR or provided herein is from CD8. In some embodiments, the hinge domain of a CAR provided herein has the sequence

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY.

[0126] In certain embodiments, a hinge or spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain includes the CH2 and/or CH3 of IgG 1, lgG4, or IgD. Illustrative spacer domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a and CD28, which may be wild-type hinge regions from these molecules or variants thereof. In certain aspects, the hinge domain includes a CD8a or CD28 hinge region. In some embodiments, the hinge is a PD-1 hinge or CD 152 hinge.

[0127] In some embodiments, the CAR further includes an extracellular spacer domain, which may include a hinge domain. The hinge domain is generally a flexible polypeptide connector region disposed between the targeting moiety and the transmembrane domain. Exemplary hinge domain sequences include those from IgG subclasses (such as IgGl and IgG4), IgD, CD28, and CD8 domains. In some embodiments, the hinge domain provides structural flexibility to flanking polypeptide regions. The hinge domain may consist of natural or synthetic polypeptides. It will be appreciated by those skilled in the art that hinge domains may improve the function of the CAR by promoting optimal positioning of the antigen binding domain in relationship to the portion of the antigen recognized by it. In some embodiments, a hinge domain may not be required for optimal CAR activity. In some embodiments, a hinge domain comprising a short sequence of amino acids promotes CAR activity by facilitating antigen-binding by, for example, relieving steric constraints that could otherwise alter antibody binding kinetics. In some embodiments, the hinge domain is linked downstream of the antigen-binding domain of a CAR and upstream of the transmembrane domain of a CAR.

[0128] Non-limiting examples of suitable hinge domains include those derived from CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4. In some embodiments, the hinge domain can include regions derived from a human CD8a (also known as CD8a) molecule, a CD28 molecule, and any other receptors that provide a similar function in providing flexibility to flanking regions. In some embodiments, the CAR disclosed herein includes a hinge domain derived from a CD8a hinge domain. In some embodiments, the CAR disclosed herein includes a hinge domain derived from a CD28 or CD8 hinge domain. In some embodiments, the hinge domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to a CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4 hinge domain.

[0129] In some embodiments, the spacer domain further comprises a linker including one or more intervening amino acid residues that are positioned between the antigen binding domain and the extracellular hinge domain. In some embodiments, the linker is positioned downstream from the antigen binding domain and upstream from the hinge domain. In principle, there are no particular limitations to the length and/or amino acid composition of the linker. In some embodiments, any arbitrary single-chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker. In some embodiments, the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues. In some embodiments, the length and amino acid composition of the extracellular spacer can be optimized to vary the orientation and/or proximity of the antigen binding domain and the extracellular hinge domain to one another to achieve a desired activity of the CAR. In some embodiments, the orientation and/or proximity of the antigen binding domain and the extracellular hinge domain to one another can be varied and/or optimized as a “tuning” tool or effect to enhance or reduce the efficacy of the CAR. In some embodiments, the orientation and/or proximity of the antigen binding domain and the hinge domain to one another can be varied and/or optimized to create a partially functional version of the CAR. In some embodiments, the extracellular spacer domain includes an amino acid sequence corresponding to an IgG4 hinge domain and an IgG4 CH2-CH3 domain.

[0130] Alternatively, the spacer domain can be a synthetic polypeptide spacer, such as a spacer having a random sequence, a (gly-gly-ser)n (“GGSn”) sequence, or a variation thereof such as (SGG)n, (GGGS)n, (SGGG)n, (GSGGG)n, and the like, where n can range from about 1 to about 15. The synthetic polypeptide spacer domain can also include a naturally occurring sequence, such as a hinge domain derived from CD8a, IgG, and the like.

[0131] The extracellular domain of the CAR is operably connected to the transmembrane domain. In some embodiments, the extracellular domain is connected to the transmembrane domain by a spacer. The transmembrane domain of the CAR serves to transduce the external signal received by the extracellular domain to the intracellular domain. The transmembrane domain can be any proper transmembrane domain known in the art, including but not limited to, CD3 transmembrane domain, CD28 transmembrane domain, CD8 transmembrane domain, CD8H transmembrane domain, and transmembrane and immunoglobulin domain containing 2 protein (CD28H). The transmembrane domain can be selected from a transmembrane region of a transmembrane protein such as, for example, Type I transmembrane proteins, an artificial hydrophobic sequence or a combination thereof. Examples of the transmembrane domain include the transmembrane regions of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Synthetic transmembrane domains may comprise a triplet of phenylalanine, tryptophan and valine. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the intracellular signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

[0132] In some embodiments, the CAR comprises a transmembrane domain from a polypeptide selected from the group consisting of: CD4, CD8a, CD28, CD 154, and PD-1; and one or more intracellular costimulatory signaling domains from a polypeptide selected from the group consisting of: 4-1BB, CD28, CD134, and CD137; and an intracellular signaling domain from a polypeptide selected from the group consisting of: FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD3zeta, CD35, CD22, CD79a, CD79, and CD665. Such a CAR may further include a spacer domain between the antigenbinding portion and the transmembrane domain, e.g., a CD8a hinge. In some embodiments, the CAR comprises a transmembrane domain from CD28. In some embodiments, the CAR comprises a transmembrane domain with the sequence FWVLVVVGGVLACYSLLVTVAFIIFWV. In some embodiments, the CAR comprises a transmembrane domain from CD8. In some embodiments, the CAR comprises a transmembrane domain with the sequence IWAPLAGTCGVLLLSLVITLYC.

[0133] The transmembrane domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, the TM domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of the T-cell receptor, CD3y, CD38, CD3E, CD35, CD3zeta, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and/or PD-1.

[0134] The transmembrane domain may include, for example without limitation, all or part of the transmembrane domain of the CD3zeta chain), CD28, CD2, CD4, 0X40, 4-1BB (CD137), ICOS (CD278), ILRB (CD122), IL-2RG (CD132), CTLA-4, PD-1, or CD40, or a sequence derived from such a transmembrane domain. The cytoplasmic signaling domain in general comprises a domain that transduces the event of ligand binding into an intracellular signal that activates the T cell. The CD3z intracellular domain/ activating domain is frequently used, although others such as MyD88 can be used. In an embodiment, the transmembrane domain is the transmembrane domain from CD3eta, CD2, CD8, or CD28. In an embodiment, the transmembrane domain is derived from the transmembrane domain from CD2 or CD28. In some embodiments, the transmembrane domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to a CD3zeta, CD28, CD2, CD4, 0X40, 4-1BB (CD137), FcERIy, ICOS (CD278), ILRB (CD122), IL-2RG (CD132), or CD40 transmembrane domain.

[0135] According to some embodiments, a CAR includes a transmembrane domain derived from CD8a or CD28 and a short polypeptide linker, e.g., between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the transmembrane domain and the intracellular signaling domain of the CAR. A glycine-serine linker may be employed as such a linker, for example.

[0136] The transmembrane domain of the CAR is operably connected to the intracellular domain. The intracellular domain serves to transduce the received external signal to kick-start the downstream signaling cascade. The intracellular domain comprises an intracellular signaling domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain from CD3 , 4-1BB (CD137) CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD35, CD3e, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70.

[0137] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from 4-1BB (CD137). In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0138] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0139] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta and an intracellular signaling domain from 4-1BB (CD137).

[0140] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD2. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPAT. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence PATSQHPPPPPGHRSQAPSHRPPPPGHRVQH.

[0141] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3epsilon. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RPPPVPNPDYEPIRKGQRDLYSGLNQRRI. In some embodiments, the CAR comprises an intracellular domain comprising a truncated CD3epsilon intracellular domain.

[0142] Signals generated through the T cell receptor (TCR) alone may be insufficient for full activation of the T cell and a secondary or costimulatory signal may also be required. Thus, T cell activation can be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR/CD3 complex) and costimulatory signaling domains that act in an antigen- independent manner to provide a secondary or costimulatory signal. As such, the CAR may include an intracellular signaling domain that includes one or more costimulatory signaling domains and a primary signaling domain.

[0143] Primary signaling domains can regulate primary activation of the TCR complex either in a stimulatory manner, or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “IT AMs”). Non-limiting examples of ITAM-containing primary signaling domains suitable for use in a CAR include those derived from FcyRI, FcRy, FcR, CD3y, CD38, CD3e, CD3zeta, CD35, CD22, CD79a, CD79b, and CD665. In certain embodiments, a CAR includes a CD3zeta primary signaling domain and one or more costimulatory signaling domains. In certain embodiments, a CAR includes a 4-1BB costimulatory signaling domain. The intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain. In certain embodiments, a CAR lacks a CD2 intracellular signaling domain.

[0144] In some embodiments, the CAR includes one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR. Exemplary costimulatory molecules suitable for use in CARs contemplated in particular embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70. In some embodiments, the costimulatory signaling domain has at least about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to a costimulatory signaling domain from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4- 1BB), CD278 (ICOS), DAP 10, LAT, KD2C, SLP76, TRIM, and/or ZAP70 domain. In some embodiments, a CAR includes one or more costimulatory signaling domains selected from the group consisting of CD2, 4-1BB, CD28, CD137, and CD134, and a CD3zeta primary signaling domain. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7- H3, and a ligand that specifically binds with CD83, and the like. In certain embodiments, the CAR comprises two or more intracellular signaling domains. For example, the CAR may comprise a first signaling domain and a second signaling domain or fragments thereof independently selected from a CD3zeta intracellular signaling domain, a CD28 intracellular signaling domain, a 4- IBB intracellular signaling domain, an OX-40 intracellular signaling domain, an inducible co-stimulator (ICOS) intracellular signaling domain, a CD27 intracellular signaling domain, and a MyD88/CD40 intracellular signaling domain. By way of example, a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof that is a CD28 intracellular signaling domain. Also, by way of example, a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof that is a 4-1BB intracellular signaling domain. Also, by way of example, a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain, a second intracellular signaling domain or fragment thereof that is a 4-1BB intracellular signaling domain, and a third intracellular signaling domain or fragment thereof that is a CD3 epsilon intracellular signaling domain.

[0145] CARs of the disclosure may comprise a CD3^, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD35, CD3s, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70 cytoplasmic signaling domain. In some embodiments, the cytoplasmic signaling domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to an CD3 , 4- 1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3E, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP 10, LAT, KD2C, SLP76, TRIM, and/or ZAP70 cytoplasmic signaling domain. CARs of the disclosure may comprise a CD2 co-stimulatory domain, and one or more additional co-stimulatory domains to increase cytokine production or sensitivity, reduce or prevent anergy, and/or to increase proliferation and cytotoxic activity. These additional co-stimulatory domains can be derived from co- stimulatory proteins such as B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7-H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 41BB (CD137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, 0X40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F-10, NTB-A, CD48, SLAM (CD 150), CD58, ikaros, CD53, integrin a4, CD82, integrin a4bl, CD90, integrin a4b7, CD96, LAG-3, CD160, LMIR, CRTAM, TCL1A, DAP12; TIM-1, Dectin-1, TIM-4, TSLP, EphB6, TSLP-R, and/or HLA- DR. In some embodiments, the cytoplasmic signaling domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to an B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7-H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 41BB (CD137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, 0X40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F-10, NTB- A, CD48, SLAM (CD150), CD58, ikaros, CD53, integrin a4, CD82, integrin a4bl, CD90, integrin a4b7, CD96, LAG-3, CD 160, LMIR, CRTAM, TCL1A, DAP 12; TIM-1, Dectin-1, TIM-4, TSLP, EphB6, TSLP-R, and/or HLA-DR domains.

[0146] In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0147] In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111. In some embodiments, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 109. In some embodiments, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 110. In some embodiments, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 111. In some embodiments, the CAR consists of an amino acid sequence of SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111. In some embodiments, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 109. In some embodiments, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 110. In some embodiments, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 111. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID Nos: 181-270. In some embodiments, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of any one of SEQ ID NOs: 181-270.

[0148] In some embodiments, the CAR further comprises a protein localization tag. The protein localization tag can be operably linked to the intracellular domain of the CAR. The protein localization tag can be operably linked to the extracellular domain of the CAR. The protein localization tag can be an ER localization tag, a Golgi apparatus (Golgi) localization tag, a lysosome localization tag, a plasma membrane localization tag, a mitochondria localization tag, a peroxisome localization tag, a cytosolic localization tag, or a nuclear localization tag. In some embodiments, the protein localization tag is an ER localization tag. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 97. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 98. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 98. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 99. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 99. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 100. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 100. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 101. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 101. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 102. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 102. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 103. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 103. In some embodiments, the ER localization tag comprises an amino acid sequence of SEQ ID NO: 104. In some embodiments, the ER localization tag consists of an amino acid sequence of SEQ ID NO: 104. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 97. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 97. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 98. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 98. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 99. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 99. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 99. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 99. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 100. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 100. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 101. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 101. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 102. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 102. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 103. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 103. In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 104. In some embodiments, the ER localization tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 104.

[0149] In some embodiments, the protein localization tag is a Golgi localization tag. In some embodiments, the Golgi localization tag comprises the amino acid sequence YQRL (SEQ ID NO: 112). In some embodiments, the Golgi localization tag consists of the amino acid sequence YQRL (SEQ ID NO: 112). In some embodiments, the protein localization tag is a lysosome localization tag. In some embodiments, the lysosome localization tag comprises the amino acid sequence KFERQ (SEQ ID NO: 113). In some embodiments, the lysosome localization tag consists of the amino acid sequence KFERQ (SEQ ID NO: 113).

[0150] In some embodiments, the ER localization tag comprising an amino acid sequence LYKYKSRRSFIDEKKMP (SEQ ID NO: 114). In some embodiments, the ER localization tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to LYKYKSRRSFIDEKKMP. In some embodiments, the ER localization tag consists of an amino acid sequence of LYKYKSRRSFIDEKKMP. In some embodiments, the ER localization tag comprises the amino acid sequence KKMP (SEQ ID NO: 115). In some embodiments, the ER localization tag consists of an amino acid sequence of KKMP.

[0151] In some embodiments, a protease cleavage site is disposed between the protein localization tag and the CAR. In some embodiments, the protease cleavage site is disposed between the protein localization tag and the intracellular domain of the CAR. In some embodiments, the protease cleavage site is disposed between the protein localization tag and the extracellular domain of the CAR. Protease cleavage sites are to be understood as amino acid residues that are recognized by proteases and/or amino acid residues whose peptide bond is cleaved by proteases. In some embodiments, a protease cleavage site can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. Optionally, additional amino acids can be present at the N-terminus and/or C-terminus of the cleavage site. A protease cleavage site also can be a variant of a cleavage site of a known protease as long as it is recognized/cleaved by the protease. [0152] Various protease cleavage sites include, but are not limited to protease cleavage sites for proteases from the serine protease family, or for metalloproteases, or for a protease from the cysteine protease family, and/or the aspartic acid protease family, and/or the glutamic acid protease family. In some embodiments, serine proteases cleavage sites include, but are not limited to, cleavage sites for chymotrypsin-like proteases, and/or subtili sin-like proteases, and/or alpha/beta hydrolases, and/or signal peptidases. In some embodiments, metalloprotease recognition sites include, but are not limited to, cleavage sites for metallocarboxypeptidases or metalloendopeptidases. In some embodiments, the protease cleavage site is TEV protease cleavage site.

[0153] The sequence encoding the split receptor switch polypeptide disclosed herein and the sequence encoding the CAR disclosed herein can be a single polynucleotide sequence. In some embodiments, the single polynucleotide sequence comprises a self-cleaving site (e.g., 2A peptides) separating the sequence encoding the split receptor switch polypeptide and the sequence encoding the CAR. In some embodiments, the sequence encoding the split receptor switch polypeptide and the sequence encoding the CAR are separated sequences. The sequence encoding the split receptor switch polypeptide or the sequence encoding the CAR can be inserted into a vector for expression in a cell. In some embodiments, the sequence encoding the split receptor switch polypeptide and the sequence encoding the CAR are inserted into the same vector. In some embodiments, the sequence encoding the split receptor switch polypeptide and the sequence encoding the CAR are inserted into different vectors. In some embodiments, the vector comprising the sequence encoding the split receptor switch polypeptide is different from the vector comprising the sequence encoding the CAR. In some embodiments, the vector comprising the sequence encoding the split receptor switch polypeptide and the vector comprising the sequence encoding the CAR are the same.

[0154] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD 19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD 19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain.

[0155] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain,

-n- and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain.

[0156] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain.

[0157] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD 19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti -CD 19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.

[0158] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain.

[0159] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD19 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain.

[0160] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain.

[0161] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain.

[0162] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a CD28 zeta intracellular signaling domain.

[0163] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL2 receptor beta, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. [0164] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL7 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain.

[0165] In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL21 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL7 receptor transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an IL2 receptor beta transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain. In some embodiments, the recombinant polynucleic acid comprises (a) the sequence encoding the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from an IL21 receptor, an UGT2B17 transmembrane domain, and an intracellular domain comprising an intracellular domain from IL9 receptor, and (b) the sequence encoding the CAR comprising an extracellular domain comprising an anti-CD22 binding domain, a CD28h or CD8h transmembrane domain, and an intracellular domain comprising a 4- IBB (CD 137) intracellular signaling domain.

[0166] In some embodiments, the recombinant polynucleic acid comprises the sequence encoding the split receptor switch polypeptide of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity of SEQ ID NOs: 1-53 and the sequence encoding the CAR of an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity of SEQ ID NOs 106-111.

[0167] The terms “nucleic acid,” “polynucleic acid” and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, and/or synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. The nucleic acid can comprise one or more bases and/or linkages that do not occur naturally in DNA or RNA, such as phosphoramidite linkages, 2’-modified ribose or deoxyribose, morpholino phosphoramidites, peptide-nucleic acid links, locked nucleic acid links, xanthine, 7-methylguanine, inosine, dihydrouracil, 5 -methylcytosine, 5- hydroxymethylcytosine, and others. See, e.g., C.I.E. Smith et al., Ann Rev Pharmacol Toxicol (2019) 59:605-30, incorporated herein by reference. A nucleic acid can be double-stranded or single-stranded (for example, a sense strand or an antisense strand). A nucleic acid may contain unconventional or modified nucleotides. The terms “polynucleotide sequence,” “polynucleic acid sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a nucleic acid molecule. [0168] In some embodiments, the recombinant polynucleic acid comprises a sequence encoding the split receptor switch polypeptide, wherein the split receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from a first cytokine receptor, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain from a second cytokine receptor; wherein the first cytokine receptor and the second cytokine receptor are different cytokine receptors. In some embodiments, the recombinant polynucleic acid comprises a sequence encoding the split receptor switch polypeptide, wherein the split receptor switch polypeptide comprises an extracellular domain comprising a cytokine binding domain from a first cytokine receptor, wherein the first cytokine receptor is an IL21 receptor, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain from a second cytokine receptor; wherein the first cytokine receptor and the second cytokine receptor are different cytokine receptors. In some embodiments, the recombinant polynucleic acid further comprises a sequence encoding a protein localization tag.

[0169] In some embodiments, the recombinant nucleic acid is operably linked to a heterologous nucleic acid sequence, such as, for example a structural gene that encodes a protein of interest or a regulatory sequence (e.g., a promoter sequence). In some embodiments, the recombinant nucleic acid is further defined as an expression cassette or a vector. In some embodiments, the vector is a lentiviral vector, an adeno virus vector, an adeno-associated virus vector, or a retroviral vector.

[0170] Some embodiments disclosed herein relate to vectors or expression cassettes including a recombinant nucleic acid molecule as disclosed herein. An expression cassette is a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell. As such, the term expression cassette may be used interchangeably with the term “expression construct.”

[0171] Also provided herein are vectors, plasmids or viruses containing one or more of the nucleic acid sequences encoding any of the split receptor switch polypeptides and the CARs disclosed herein. The nucleic acid sequences described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. Suitable vectors for use in eukaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al, Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al, “Molecular Cloning: A Laboratory Manual,” 2nd Ed. (1989).

Delivery Vectors [0172] Accordingly, in some embodiments, the split receptor switch polypeptides and the CARs of the present disclosure can be expressed from vectors, generally expression vectors. The vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non- episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno- associated viruses) are also included.

[0173] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1 89) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0174] Vectors suitable for use include the pMSXND expression vector for use in mammalian cells. In some embodiments, nucleic acid inserts, which encode the split receptor switch polypeptides and/or the CARs in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought. Viral vectors that can be used in the disclosure include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0175] In some embodiments, the expression vector is a viral vector. The term “viral vector” is widely used to refer either to a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell, or to a viral particle that mediates nucleic acid transfer. Viral particles typically include viral components, and sometimes also host cell components, in addition to nucleic acid(s). Retroviral vectors used herein contain structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. Retroviral lentivirus vectors contain structural and functional genetic elements, or portions thereof including LTRs, that are primarily derived from a lentivirus (a sub-type of retrovirus). [0176] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors (including lentivirus vectors), adenovirus vectors, and adeno-associated virus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0177] In some embodiments, the nucleic acid molecules are delivered by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for stable or transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can also be accomplished using classical random genomic recombination techniques or with more precise genome editing techniques such as using guide RNA-directed CRISPR/Cas9, DNA-guided endonuclease genome editing NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a mini circle expression vector for stable or transient expression.

[0178] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle. Alternatively, endonuclease polypeptide(s) can be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. For example, introduction of nucleic acids into cells may be achieved using viral transduction methods. In a non-limiting example, adeno- associated virus (AAV) is a non-enveloped virus that can be engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0179] Lentiviral systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into the host cell genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy -target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile (e.g., by targeting a site for integration that has little or no oncogenic potential); and (vii) a relatively easy system for vector manipulation and production.

[0180] In some embodiments, the sequence encoding the CAR and the sequence encoding the first, second, third, and/or fourth polypeptides is delivered in a single vector. In some embodiments, the sequence encoding the CAR and the sequence encoding the first, second, third, and/or fourth polypeptides is delivered in two or more vectors. In some embodiments, each of the first, second, third, and/or fourth polypeptides further comprises a CAR, and each of the first, second, third, and/or fourth polypeptides further comprising a CAR are delivered to a cell in a separate vector.

Compositions [0181] In other aspects, the present disclosure describes compositions comprising any of the split receptor switch polypeptides disclosed herein. In another aspect, the present disclosure provides compositions comprising any of the recombinant polynucleic acids disclosed herein. In some embodiments, the composition comprising the split receptor switch polypeptides disclosed herein and the CAR disclosed herein. In some embodiments, the composition comprising the recombinant polynucleic acids encoding the split receptor switch polypeptides disclosed herein and the CAR disclosed herein.

[0182] In some embodiments, the recombinant polynucleic acid comprising the split receptor switch polypeptide comprises a first recombinant polynucleic acid molecule encoding the first polypeptide, a second recombinant polynucleic acid molecule encoding the second polypeptide, a third recombinant polynucleic acid molecule encoding the third polypeptide, and/or a fourth recombinant polynucleic acid molecule encoding the fourth polypeptide.

Cells

[0183] In some aspects, the present disclosure describes cells comprising the split receptor switch polypeptides disclosed herein, cells comprising the recombinant polynucleic acids disclosed herein, cells comprising the compositions comprising the split receptor switch polypeptides disclosed herein, and cells comprising the compositions comprising the recombinant polynucleic acids disclosed herein. In some embodiments, the cell expresses the split receptor switch polypeptides disclosed herein. In some embodiments, the split receptor switch polypeptide is expressed on the surface of the cell. By “expressed on the surface of the cell” or “cell surface expression” is meant the cell surface molecule - when no longer associated with the protein localization tag (e.g., ER localization tag, Golgi localization tag, or the like) has been trafficked to the cell membrane such that - in the case of a cell surface receptor (e.g., a CAR, TCR, etc.) - the extracellular binding domain is displayed on the cell surface, the transmembrane portion passes through the cell membrane, and the one or more intracellular signaling domains are disposed adjacent to the intracellular side of the cell membrane. Upon binding of the extracellular binding domain to the target ligand/antigen, the intracellular signaling domain of the cell surface receptor participates in transducing the signal from the binding into the interior of the cell.

[0184] In some embodiments, host cells can be genetically engineered (e.g transduced, transformed, or transfected) with, for example, a vector construct of the present disclosure that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule. In some embodiments, the host cell is an immune cell, a stem cell, a mammalian cell, a primate cell, or a human cell. In some embodiments, the host cell is autologous or allogeneic. In some embodiments, the host cell is a T cell, a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, or a tumor infiltrating lymphocyte.

[0185] Host cells can be transduced with a nucleic acid encoding the split receptor switch polypeptide and/or a CAR, with or without the protein localization tag. In some embodiments, a host cell can be transduced with a nucleic acid encoding a split receptor switch polypeptide. In some embodiments, a host cell can be transduced with a bicistronic nucleic acid encoding a split receptor switch polypeptide and a CAR. In some embodiments, a host cell can be transduced with a nucleic acid encoding a split receptor switch polypeptide and an additional nucleic acid encoding a CAR. In some embodiments, a host cell can be transduced with a bicistronic nucleic acid encoding a split receptor switch polypeptide with a protein localization tag and a CAR. In some embodiments, a host cell can be transduced with a bicistronic nucleic acid encoding a split receptor switch polypeptide and a CAR with a protein localization tag. In some embodiments, a host cell can be transduced with a bicistronic nucleic acid encoding a split receptor switch polypeptide with a protein localization tag and a CAR with another protein localization tag. In some embodiments, the host cell is further transduced with an additional nucleic acid encoding one or more additional therapeutic agents such as, for example, but not limited to, an antibody, an antibody fragment thereof, or a protein therapeutic.

[0186] In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a mouse cell. In some embodiments, the animal cell is a human cell. In some embodiments, the recombinant cell is an immune system cell, e.g., a lymphocyte (for example without limitation, a T cell, natural killer cell or NK cell, natural killer T cell or NKT cell, a B cell, a plasma cell, tumor-infiltrating lymphocyte (TIL)), a monocyte or macrophage, or a dendritic cell. In some embodiments, the immune system cell is selected from the group consisting of B cells, T cells, monocytes, dendritic cells, and epithelial cells. In some embodiments, the immune system cell is a T lymphocyte. The immune cell can also be a precursor cell, i.e., a cell that is capable of differentiating into an immune cell.

[0187] Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. In some embodiments, the nucleic acid molecule is introduced into a host cell by a transduction procedure, electroporation procedure, or a biolistic procedure. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0188] Cells of the present disclosure may be autologous/autogeneic (“self’) or non-autologous (“nonself,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells derived from the same individual to which they are subsequently administered. “Allogeneic” as used herein refers to cells of the same species that differ genetically from the cell in comparison. “Syngeneic,” as used herein, refers to cells of a different individual that are genetically identical to the cell in comparison. In some embodiments, the cells are T cells obtained from a mammal. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human.

[0189] T cells can be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells are obtained from a unit of blood collected from an individual using any number of known techniques such as sedimentation, e.g., FICOLL™ separation.

[0190] In some embodiments, an isolated or purified population of T cells is used. In some embodiments, TCTL and TH lymphocytes are purified from PBMCs. In some embodiments, the TCTL and TH lymphocytes are sorted into naive (TN), memory (TMEM), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and effector (TEFF) T cell subpopulations either before or after activation, expansion, and/or genetic modification. Suitable approaches for such sorting are known and include, e.g., magnetic-activated cell sorting (MACS), where TN are CD45RA+ CD62L+ CD95-; TSCM are CD45RA+ CD62L+ CD95+; TCM are CD45RO+ CD62L+ CD95+; and TEM are CD45RO+ CD62L- CD95+. An exemplary approach for such sorting is described in Wang et al. (2016) Blood 127(24):2980- 90.

[0191] A specific subpopulation of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection techniques. In some embodiments, a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38, is further isolated by positive or negative selection techniques. In some embodiments, the manufactured T cell compositions do not express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3. In some embodiments, the manufactured T cell compositions do not substantially express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3.

[0192] Further disclosed herein includes a composition comprising a cell comprising a recombinant polynucleic acid comprising a sequence encoding a split receptor switch polypeptide, wherein the cell expresses the split receptor switch polypeptide, the split receptor switch polypeptide comprising an extracellular domain comprising a cytokine binding domain from a first cytokine receptor, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain from a second cytokine receptor, wherein the first cytokine receptor and the second cytokine receptor are different cytokine receptors; and wherein the cell expands at least 2-fold greater in the presence of a cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 3 -fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 4-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 5-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 10-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 15-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 20-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 30-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 40-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 50-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 100-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 200-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 300-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 400-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 500-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 700-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 1000-fold greater in the presence of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine.

[0193] In some embodiments, the cell expands at least 2-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 3-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 4-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 5-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 10-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 20-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 30-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 40-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 50-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 100-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 200-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 300-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 400-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 500-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine.

[0194] In some embodiments, the cell expands at least 2-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 10-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 50-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 100-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 200-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 500-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 1000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 2000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 3000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 4000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the cell expands at least 5000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain compared to a cell that does not express the split receptor switch polypeptide in the presence of the cytokine.

[0195] In some embodiments, the cell is a mammalian cell. The cell can be a human cell. The cell can be a blood cell. In some embodiments, the blood cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the cell is a population of cells. In some embodiments, the population of cells is a population of blood cells. The blood cells can be lymphocytes. The lymphocytes can be T cells. In some embodiments, the population of cells is a homogeneous mixture of cells of the same cell type. In some embodiments, the population of cells is a heterogeneous mixture of cells of different cell types. In some embodiments, the population of cells comprises at least about IxlO ⁵ cells. In some embodiments, the population of cells comprises at least about IxlO ⁶ cells. In some embodiments, the population of cells comprises at least about IxlO ⁷ cells. In some embodiments, the population of cells comprises at least about IxlO ⁸ cells. In some embodiments, the population of cells comprises at least about IxlO ⁹ cells. In some embodiments, the population of cells comprises from about IxlO ⁵ cells to about IxlO ⁹ cells. In some embodiments, the population of cells comprises from about IxlO ⁵ cells to about IxlO ⁸ cells. In some embodiments, the population of cells comprises from about IxlO ⁵ cells to about IxlO ⁷ cells. In some embodiments, the population of cells comprises from about IxlO ⁵ cells to about IxlO ⁶ cells. In some embodiments, at least 50% or more of the cells in the population of cells express at least two CARs. In some embodiments, at least 60% or more of the cells in the population of cells express at least two CARs. In some embodiments, at least 65% or more of the cells in the population of cells express at least two CARs. In some embodiments, at least 50% or more of the cells in the population of cells express at least three CARs. In some embodiments, at least 60% or more of the cells in the population of cells express at least three CARs. In some embodiments, at least 65% or more of the cells in the population of cells express at least three CARs.

[0196] In some embodiments, addition of IL2 can stimulate and expand both the cell expressing the split receptor switch polypeptide and the cell not expressing the split receptor switch polypeptide. Specifically, the cell that expresses the split receptor switch polypeptide expands in the presence of IL2 and a cell that does not express the split receptor switch polypeptide expands in the presence of IL2.

[0197] In some embodiments, addition of the cytokine that specifically binds to the extracellular domain of the split receptor switch polypeptide can stimulate and expand only the cell expressing the split receptor switch polypeptide but not the cell not expressing the split receptor switch polypeptide. Specifically, the cell that does not express the split receptor switch polypeptide does not expand in the presence of the cytokine that binds to the extracellular domain of the split receptor switch polypeptide. In some embodiments, the cytokine is IL21.

Pharmaceutical Compositions

[0198] In some aspects, the present disclosure describes a pharmaceutical composition comprising the composition comprising the split receptor switch polypeptides disclosed herein, and a pharmaceutically acceptable excipient or carrier. In some aspects, the present disclosure describes a pharmaceutical composition comprising the compositions comprising the recombinant polynucleic acids disclosed herein, and a pharmaceutically acceptable excipient or carrier. In some aspects, the present disclosure describes a pharmaceutical composition comprising the composition comprising the cells disclosed herein, and a pharmaceutically acceptable excipient or carrier. The pharmaceutical compositions generally include a therapeutically effective amount of the cells. By “therapeutically effective amount” is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease (e.g., cancer) or disorder associated, e.g., with the target cell or a population thereof (e.g., cancer cells), as compared to a control. An effective amount can be administered in one or more administrations. A “therapeutically effective amount” of the cells disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” an individual, e.g., a patient. When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual). In some embodiments, a pharmaceutical composition of the present disclosure includes from IxlO ⁵ to 5xlO ¹⁰ of the cells of the present disclosure.

[0199] The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents.

[0200] Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration. [0201] The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.

[0202] Pharmaceutical compositions that include the cells of the present disclosure may be prepared by mixing the cells having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m- cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0203] An aqueous formulation of the recombinant polypeptides, proteases, nucleic acids, expression vectors, and/or cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0204] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0205] A surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylenepolypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w/v.

[0206] In some embodiments, the pharmaceutical composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).

Methods of Treatment

[0207] Further disclosed here include methods for treating a disease, disorder, or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein. The pharmaceutical composition can be administered alone or in combination with other agents (e.g., an antibody or an antigen binding fragment thereof, or a molecule). In some embodiments, a vaccine, an oncoloytic viruse, a checkpoint inhibitor, a T cell agonist antibody, chemotherapy, and/or a bispecific antibody can be combined with the pharmaceutical composition disclosed herein. In some embodiments, the pharmaceutical composition is administered with other cells (e.g., CAR T cells or other adoptively transferred T cells). Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. In some embodiments, the one or more additional therapeutic agents, chemotherapeutics, anti-cancer agents, or anti-cancer therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. “Chemotherapy” and “anti-cancer agent” are used interchangeably herein. Various classes of anti-cancer agents can be used. Non-limiting examples include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), checkpoint inhibitors, immunomodulators, cytokines, nanoparticles, radiation therapy, tyrosine kinase inhibitors (for example, imatinib mesylate), hormone treatments, soluble receptors and other antineoplastics. [0208] In some embodiments, a method of treating a disease, disorder, or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein and administering a therapeutically effective amount of IL-21.

[0209] In some embodiments, the disease, disorder, or condition is a cancer, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, or an autoimmune disease. In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the disease, disorder, or condition is a hyperproliferative disorder. Hyperproliferative disorders include cancers and hyperplasia characterized by the unregulated overgrowth of cells. Hyperproliferative disorders frequently display loss of genetic regulatory mechanisms, and may express native proteins inappropriately (including expression of proteins from other cell types or developmental stages, expression of mutated proteins, and expression of proteins at levels higher or lower than normal).

[0210] B-cell hyperproliferative disorders include B-cell leukemias and lymphomas such as, but not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia, diffuse large B- cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, MALT lymphoma, Waldenstrom’s macroglobulinemia, and/or other disorders characterized by the overgrowth of B-lineage cells.

[0211] Hyperproliferative disorders include diseases such as, but not limited to, bladder cancer, including upper tract tumors and urothelial carcinoma of the prostate; bone cancer, including chondrosarcoma, Ewing's sarcoma, and osteosarcoma; breast cancer, including noninvasive, invasive, phyllodes tumor, Paget's disease, and breast cancer during pregnancy; central nervous system cancers, adult low-grade infiltrative supratentorial astrocytoma/oligodendroglioma, adult intracranial ependymoma, anaplastic astrocytoma/anaplastic oligodendroglioma/glioblastoma multiforme, limited (1-3) metastatic lesions, multiple (>3) metastatic lesions, carcinomatous lymphomatous meningitis, non-immunosuppressed primary CNS lymphoma, and metastatic spine tumors; cervical cancer; colon cancer, rectal cancer, anal carcinoma; esophageal cancer; gastric (stomach) cancer; head and neck cancers, including ethmoid sinus tumors, maxillary sinus tumors, salivary gland tumors, cancer of the lip, cancer of the oral cavity, cancer of the oropharynx, cancer of the hypopharynx, occult primary, cancer of the glottic larynx, cancer of the supraglottic larynx, cancer of the nasopharynx, and advanced head and neck cancer; hepatobiliary cancers, including hepatocellular carcinoma, gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma; Hodgkin disease/lymphoma; kidney cancer; melanoma; multiple myeloma, systemic light chain amyloidosis, Waldenstrom's macroglobulinemia; myelodysplastic syndromes; neuroendocrine tumors, including multiple endocrine neoplasia, type 1, multiple endocrine neoplasia, type 2, carcinoid tumors, islet cell tumors, pheochromocytoma, poorly differentiated/small cell/atypical lung carcinoids; Non-Hodgkin's Lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B-Cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, AIDS-Related B-Cell lymphoma, peripheral T-Cell lymphoma, and mycosis fungoides/Sezary Syndrome; non-melanoma skin cancers, including basal and squamous cell skin cancers, dermatofibrosarcoma protuberans, Merkel cell carcinoma; non-small cell lung cancer (NSCLC), including thymic malignancies; occult primary; ovarian cancer, including epithelial ovarian cancer, borderline epithelial ovarian cancer (Low Malignant Potential), and less common ovarian histologies; pancreatic adenocarcinoma; prostate cancer; small cell lung cancer and lung neuroendocrine tumors; soft tissue sarcoma, including soft-tissue extremity, retroperitoneal, intraabdominal sarcoma, and desmoid; testicular cancer; thymic malignancies, including thyroid carcinoma, nodule evaluation, papillary carcinoma, follicular carcinoma, Hurthle cell neoplasm, medullary carcinoma, and anaplastic carcinoma; uterine neoplasms, including endometrial cancer and/or uterine sarcoma.

[0212] Methods for administering immune cells for therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described in US 2003/0170238; US 4690915; S.A. Rosenberg, Nat Rev Clin Oncol (2011) 8(10):577- 85. See also M. Themeli et al., Nat Biotechnol (2013) 31 (10):928-33 ; and T. Tsukahara et al., Biochem Biophys Res Commun (2013) 438(1): 84-89.

Methods for Expanding Cells

[0213] Further disclosed herein include methods for expanding cells, comprising culturing the cell disclosed herein in the presence of the cytokine that binds to the extracellular domain of the split receptor switch polypeptide, thereby expanding the cell. In some embodiments, the cell is expanded at least 2-fold. In some embodiments, the cell is expanded at least 3-fold. In some embodiments, the cell is expanded at least 4-fold. In some embodiments, the cell is expanded at least 5-fold. In some embodiments, the cell is expanded at least 10-fold. In some embodiments, the cell is expanded at least 20-fold. In some embodiments, the cell is expanded at least 30-fold. In some embodiments, the cell is expanded at least 40-fold. In some embodiments, the cell is expanded at least 50-fold. In some embodiments, the cell is expanded at least 100-fold. In some embodiments, the cell is expanded at least 200-fold. In some embodiments, the cell is expanded at least 300-fold. In some embodiments, the cell is expanded at least 400-fold. In some embodiments, the cell is expanded at least 500-fold. In some embodiments, the cell is expanded at least 1000-fold. [0214] In some embodiments, the cell is expanded a least 2-fold on day 10 after addition of the cytokine that binds to the extracellular domain of the split receptor switch polypeptide. In some embodiments, the cell is expanded at least 3-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 4-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 5-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 10-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 20-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 30-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 40-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 50-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 100-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 200-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 300-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 400-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 500-fold on day 10 after addition of the cytokine. In some embodiments, the cell is expanded at least 1000-fold on day 10 after addition of the cytokine.

[0215] In some embodiments, the cell is expanded at least 2-fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 3 -fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 4-fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 5-fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 10-fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 50-fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 100-fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 500-fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 1000-fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 1500-fold on day 14 after addition of the cytokine. In some embodiments, the cell is expanded at least 2000-fold on day 14 after addition of the cytokine.

Methods for Preparing CAR-Expressing Immune Effector Cells

[0216] Further disclosed herein include methods for making a population of CAR-expressing immune effector cells, comprising contacting the cell disclosed herein with a cytokine that binds to the cytokine binding domain from the first cytokine receptor, and expanding the cell in the presence of the cytokine that binds to the cytokine binding domain from the first cytokine receptor, thereby making a population of CAR-expressing immune effector cells. [0217] In some embodiments, the first cytokine receptor is IL21 receptor. In some embodiments, the cytokine is IL21. In some embodiments, the first cytokine receptor is IL21 receptor and the cytokine is IL21. IL21 can be added to make a population of the CAR-expressing immune effector cells expressing the split receptor switch polypeptide. In some embodiments, the CAR-expressing immune effector cells, after culturing with the cytokine, for example IL21, are expanded at least 2-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 3-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 4-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 5-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 10-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 20-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 30-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 40-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 50-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 100-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 200-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 300-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 400-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 500-fold. In some embodiments, the CAR-expressing immune effector cells are expanded at least 1000-fold.

[0218] In some embodiments, the CAR-expressing immune effector cells are expanded a least 2-fold on day 10 after addition of the cytokine that binds to the extracellular domain of the split receptor switch polypeptide, for example, IL21. In some embodiments, the CAR-expressing immune effector cells are expanded at least 3-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 4-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 5- fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 10-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 20-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 30-fold on day 10 after addition of the cytokine. In some embodiments, the CAR- expressing immune effector cells are expanded at least 40-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 50-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 100-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 200-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 300- fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 400-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 500-fold on day 10 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 1000-fold on day 10 after addition of the cytokine.

[0219] In some embodiments, the CAR-expressing immune effector cells are expanded at least 2-fold on day 14 after addition of the cytokine (e.g., IL21). In some embodiments, the CAR-expressing immune effector cells are expanded at least 3-fold on day 14 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 4-fold on day 14 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 5-fold on day 14 after addition of the cytokine. In some embodiments, the CAR- expressing immune effector cells are expanded at least 10-fold on day 14 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 50-fold on day 14 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 100-fold on day 14 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 500-fold on day 14 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 1000-fold on day 14 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 1500-fold on day 14 after addition of the cytokine. In some embodiments, the CAR-expressing immune effector cells are expanded at least 2000-fold on day 14 after addition of the cytokine.

[0220] In some embodiments, the CAR-expressing immune effector cells expands at least 2-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 3-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 4-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 5-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 10-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR- expressing immune effector cells expands at least 15-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 20-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR- expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 30- fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 40-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 50-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 100-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR- expressing immune effector cells expands at least 200-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 300-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR- expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 400- fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 500-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 700-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cells expands at least 1000-fold greater in the presence of a cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune cell that does not express the split receptor switch polypeptide in the presence of the cytokine.

[0221] In some embodiments, the CAR-expressing immune effector cell expands at least 2-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 3 -fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR- expressing immune effector cell expands at least 4-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 5-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR- expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 10-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 20-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 30-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR- expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 40-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 50-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 100-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR- expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 200-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 300-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 400-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR- expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 500-fold greater on day 10 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine.

[0222] In some embodiments, the CAR-expressing immune effector cell expands at least 2-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 10-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR- expressing immune effector cell expands at least 50-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 100-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 200-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR- expressing immune effector cell expands at least 500-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 1000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 2000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR- expressing immune effector cell expands at least 3000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 4000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the CAR-expressing immune effector cell expands at least 5000-fold greater on day 14 after addition of the cytokine that binds to the extracellular domain (e.g., IL21) compared to a CAR-expressing immune effector cell that does not express the split receptor switch polypeptide in the presence of the cytokine. In some embodiments, the immune effector cell is a mammalian cell. The cell can be a human cell. In some embodiments, the immune effector cell is a lymphocyte. In some embodiments, the lymphocyte is a T cell. In some embodiments, the cell is a population of cells. In some embodiments, the population of cells is a homogeneous mixture of cells of the same cell type. In some embodiments, the population of cells is a heterogeneous mixture of cells of different cell types. In some embodiments, the population of cells comprises at least about IxlO ⁵ cells. In some embodiments, the population of cells comprises at least about IxlO ⁶ cells. In some embodiments, the population of cells comprises at least about IxlO ⁷ cells. In some embodiments, the population of cells comprises at least about IxlO ⁸ cells. In some embodiments, the population of cells comprises at least about IxlO ⁹ cells. In some embodiments, the population of cells comprises from about IxlO ⁵ cells to about IxlO ⁹ cells, In some embodiments, the population of cells comprises from about IxlO ⁵ cells to about IxlO ⁸ cells, In some embodiments, the population of cells comprises from about IxlO ⁵ cells to about IxlO ⁷ cells, In some embodiments, the population of cells comprises from about IxlO ⁵ cells to about IxlO ⁶ cells.

[0223] In some embodiments, using the methods described herein a higher percentage of the cells can be obtained that express both a first protein of interest (e.g., a first CAR) encoded by a first vector, and a second protein of interest (e.g., a second CAR) encoded by a second vector compared to conventional methods known in the art. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding a first portion of a split receptor switch polypeptide, and a second vector having a sequence encoding a second protein of interest (e.g., a second CAR) and a sequence encoding a second portion of a split receptor switch polypeptide followed by incubation of the cells in the presence of a cytokine that binds to the first portion of a split receptor switch polypeptide can produce a population of cells in which the percentage of the cells in the population that express both the first protein of interest (e.g., a first CAR) and the second protein of interest (e.g., a second CAR) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or higher. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding a first portion of a split receptor switch polypeptide, and a second vector having a sequence encoding a second protein of interest (e.g., a second CAR) and a sequence encoding a second portion of a split receptor switch polypeptide followed by incubation of the cells in the presence of a cytokine that binds to the first portion of a split receptor switch polypeptide can produce a population of cells in which a higher percentage of the cells in the population express both the first protein of interest (e.g., a first CAR) and the second protein of interest (e.g., a second CAR) compared to the percentage of the cells that express both the first protein of interest (e.g., a first CAR) and the second protein of interest (e.g., a second CAR) when the cell population is transduced with a first vector encoding the first protein of interest (e.g., a first CAR) but without the sequence encoding the first portion of the split receptor switch polypeptide, and a second vector encoding the second protein of interest (e.g., a second CAR) but without the sequence encoding the second portion of the split receptor switch polypeptide. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding a first portion of a split receptor switch polypeptide, and a second vector having a sequence encoding a second protein of interest (e.g., a second CAR) and a sequence encoding a second portion of a split receptor switch polypeptide followed by incubation of the cells in the presence of a cytokine that binds to the first portion of a split receptor switch polypeptide can produce a population of cells in which the percentage of the cells in the population that express both the first protein of interest (e.g., a first CAR) and the second protein of interest (e.g., a second CAR) is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,

2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50-fold higher than the percentage of the cells that express both the first protein of interest (e.g., a first CAR) and the second protein of interest (e.g., a second CAR) when the cell population is transduced with a first vector encoding the first protein of interest (e.g., a first CAR) but without the sequence encoding the first portion of the split receptor switch polypeptide, and a second vector encoding the second protein of interest (e.g., a second CAR) but without the sequence encoding the second portion of the split receptor switch polypeptide.

[0224] In some embodiments, using the methods described herein a higher percentage of the cells can be obtained that express each of a first protein of interest (e.g., a first CAR) encoded by a first vector, and a second protein of interest (e.g., a second CAR) and a third protein of interest (e.g., a third CAR) encoded by the second vector compared to conventional methods known in the art. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding a first portion of a split receptor switch polypeptide, and a second vector having a sequence encoding a second protein of interest (e.g., a second CAR), a sequence encoding a third protein of interest (e.g., a third CAR) and a sequence encoding a second portion of a split receptor switch polypeptide followed by incubation of the cells in the presence of a cytokine that binds to the first portion of a split receptor switch polypeptide can produce a population of cells in which the percentage of the cells in the population that express both the first protein of interest (e.g., a first CAR), the second protein of interest (e.g., a second CAR) and the third protein of interest (e.g., a third CAR) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or higher. For example, transduction of a population of cells with a first vector having a sequence encoding the first protein of interest (e.g., a first CAR) and a sequence encoding a first portion of a split receptor switch polypeptide, and a second vector having a sequence encoding a second protein of interest (e.g., a second CAR), a sequence encoding a third protein of interest (e.g., a third CAR) and a sequence encoding a second portion of a split receptor switch polypeptide followed by incubation of the cells in the presence of a cytokine that binds to the first portion of a split receptor switch polypeptide can produce a population of cells in which the percentage of the cells in the population that express each of the first protein of interest (e.g., a first CAR), the second protein of interest (e.g., a second CAR) and the third protein of interest (e.g., a third CAR) is at least 1.1, 1.2,

1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50-fold higher than the percentage of the cells that express each of the first protein of interest (e.g., a first CAR), the second protein of interest (e.g., a second CAR) and the third protein of interest (e.g., a third CAR) when the cell population is transduced with a first vector encoding the first protein of interest (e.g., a first CAR) but without the sequence encoding the first portion of the split receptor switch polypeptide, and a second vector encoding the second protein of interest (e.g., a second CAR) and the third protein of interest (e.g., a third CAR) but without the sequence encoding the second portion of the split receptor switch polypeptide.

[0225] Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0226] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dentritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes.

List of Numbered Embodiments

1. A composition comprising a recombinant polynucleic acid comprising a first sequence encoding a first polypeptide comprising a first portion of a split receptor switch polypeptide and a second sequence encoding a second polypeptide comprising a second portion of the split receptor switch polypeptide, wherein the first polypeptide comprises:

(a) a first extracellular domain comprising a cytokine binding domain from a first cytokine receptor, and

(b) a first transmembrane domain, wherein the second polypeptide comprises: (a) a second transmembrane domain, and

(b) a first intracellular domain comprising an intracellular signaling domain from a second cytokine receptor; wherein the first cytokine receptor and the second cytokine receptor are different cytokine receptors. The composition of embodiment 1, wherein the first cytokine receptor is an IL9 or IL21 receptor. The composition of embodiment 1, wherein the first cytokine receptor is an IL-2RG (CD132) receptor. A composition comprising a recombinant polynucleic acid comprising a first sequence encoding a first polypeptide comprising a first portion of a split receptor switch polypeptide and a second sequence encoding a second polypeptide comprising a second portion of the split receptor switch polypeptide, wherein the first polypeptide comprises:

(a) a first extracellular domain comprising a cytokine binding domain from a first cytokine receptor, and

(b) a first transmembrane domain, wherein the second polypeptide comprises:

(a) a second transmembrane domain, and

(b) a first intracellular domain comprising an intracellular signaling domain from the first cytokine receptor. The composition of embodiment 4, wherein the first cytokine receptor is an IL-2RG (CD132) receptor. The composition of embodiment 1, wherein the first polypeptide and the second polypeptide form a complex comprising the split receptor switch polypeptide. The composition of embodiment 1, wherein the first polypeptide and the second polypeptide form a complex. The composition of embodiment 1, wherein the split receptor switch polypeptide is a functional split receptor switch polypeptide. The composition of embodiment 1, wherein the first transmembrane domain interacts with the second transmembrane domain. The composition of embodiment 1, wherein the first transmembrane domain and the second transmembrane domain form a multimer. The composition of embodiment 10, wherein the first transmembrane domain and the second transmembrane domain form a homodimer or a heterodimer. The composition of embodiment 1, wherein the second polypeptide is not a functional split receptor switch polypeptide A composition comprising a cell comprising the composition of embodiment 1, wherein the cell expresses the split receptor switch polypeptide. The composition of embodiment 13, wherein the split receptor switch polypeptide is a functional split receptor switch polypeptide capable of signaling to the cell. The composition of embodiment 13, wherein the first polypeptide is not a functional split receptor switch polypeptide capable of signaling to the cell. The composition of embodiment 13, wherein the second polypeptide is not a functional split receptor switch polypeptide capable of signaling to the cell. The composition of embodiment 13, wherein the first intracellular signaling domain is activated in the presence of a cytokine that binds to the extracellular domain of the first polypeptide. The composition of embodiment 13, wherein the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide in a cell that expresses both the first and second polypeptide compared to a cell that expresses only the first polypeptide, only the second polypeptide or neither the first polypeptide nor the second polypeptide. The composition of embodiment 13, wherein the cell expands at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that expresses only the first polypeptide, only the second polypeptide, or neither the first polypeptide nor the second polypeptide. The composition of embodiment 13, wherein the cell expresses endogenous IL-2RG (CD132) receptor. The composition of embodiment 20, wherein the endogenous IL-2RG (CD132) receptor forms a complex with the first polypeptide, the second polypeptide or both. The composition of embodiment 20, wherein the first polypeptide, the second polypeptide and the endogenous IL-2RG (CD 132) receptor form a complex comprising the split receptor switch polypeptide. The composition of embodiment 20, wherein the first polypeptide, the second polypeptide and the endogenous IL-2RG (CD 132) receptor form a complex comprising the split receptor switch polypeptide in the presence of a cytokine. The composition of embodiment 23, wherein the first polypeptide, the second polypeptide and the endogenous IL-2RG (CD 132) receptor do not form a complex comprising the split receptor switch polypeptide in the absence of a cytokine. The composition of embodiment 13, wherein the cell does not express endogenous IL-2RG (CD132) receptor. The composition of embodiment 25, wherein the cell expresses an exogenous IL-2RG (CD132). The composition of embodiment 25, wherein the composition further comprises a recombinant polynucleic acid comprising a sequence encoding IL-2RG (CD132). The composition of any one of embodiments 13-27, wherein the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that does not express a IL-2RG (CD 132) receptor. The composition of any one of embodiments 1-28, wherein the first polypeptide further comprises an intracellular domain from a third cytokine receptor. The composition of embodiment 29, wherein the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor and the second cytokine receptor. The composition of any one of embodiments 1-30, wherein the second polypeptide further comprises a second extracellular domain from a third cytokine receptor. The composition of embodiment 31, wherein the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor and the second cytokine receptor. The composition of embodiment 31, wherein the first extracellular domain of the first polypeptide comprises a first portion of the cytokine binding domain, and wherein the second extracellular domain of the third polypeptide comprises a second portion of the cytokine binding domain. The composition of any one of embodiments 1-33, wherein the third cytokine receptor is an IL-2RG (CD 132) receptor. The composition of any one of embodiments 1-34, wherein the recombinant polynucleic acid comprises a first recombinant polynucleic acid molecule comprising the first sequence and a second recombinant polynucleic acid molecule comprising the second sequence. The composition of embodiment 35, wherein the first recombinant polynucleic acid further comprises a sequence encoding a first gene of interest. The composition of embodiment 36, wherein the sequence encoding a first gene of interest gene of interest encodes for a first protein of interest, wherein the first polypeptide and the first protein of interest are expressed as a single polypeptide molecule. The composition of embodiment 37, wherein the sequence encoding the first polypeptide is linked to the encoding the first protein of interest by a sequence encoding a linker. The composition of embodiment 38, wherein the linker comprises a self-cleaving peptide sequence, a protease cleavage site or an IRES. The composition of any one of embodiments 35-39, wherein the second recombinant polynucleic acid further comprises a sequence encoding a second gene of interest. The composition of embodiment 40, wherein the sequence encoding a second gene of interest gene of interest encodes for a second protein of interest, wherein the second polypeptide and the second protein of interest are expressed as a single polypeptide molecule. The composition of embodiment 41, wherein the sequence encoding the second polypeptide is linked to the encoding the second protein of interest by a sequence encoding a linker. The composition of embodiment 42, wherein the linker comprises a self-cleaving peptide sequence, a protease cleavage site or an IRES. The composition of any one of embodiments 35-42, wherein the recombinant polynucleic acid comprises a third recombinant polynucleic acid comprising a sequence encoding a third polypeptide. The composition of any one of embodiments 1-35, wherein the first sequence and the second sequence are not comprised within the same recombinant polynucleic acid molecule. The composition of any one of embodiments 1-45, wherein the recombinant polynucleic acid further comprises a third sequence encoding a third polypeptide comprising a third portion of the split receptor switch polypeptide. The composition of embodiment 46, wherein the third recombinant polynucleic acid further comprises a sequence encoding a third gene of interest. The composition of embodiment 47, wherein the sequence encoding a third gene of interest encodes for a third protein of interest, wherein the third polypeptide and the third protein of interest are expressed as a single polypeptide molecule. The composition of embodiment 48, wherein the sequence encoding the second polypeptide is linked to the encoding the second protein of interest by a sequence encoding a linker. The composition of embodiment 49, wherein the linker comprises a self-cleaving peptide sequence, a protease cleavage site or an IRES. The composition of any one of embodiments 46-50, wherein the third polypeptide comprises: a. a third transmembrane domain, and b. a second intracellular domain comprising an intracellular domain from a third cytokine receptor. The composition of embodiment 51, wherein the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor. The composition of embodiment 51 or 52, wherein the third cytokine receptor is a cytokine receptor that is different than the second cytokine receptor. The composition of any one of embodiments 46-50, wherein the third polypeptide comprises: a. a second extracellular domain from a third cytokine receptor; and b. a third transmembrane domain. The composition of embodiment 54, wherein the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor. The composition of embodiment 54 or 55, wherein the third cytokine receptor is a cytokine receptor that is different than the second cytokine receptor. The composition of any one of embodiments 46-50, wherein the third polypeptide comprises: a. a second extracellular domain from a third cytokine receptor, wherein the third cytokine receptor is a cytokine receptor that is different than the first cytokine receptor and the second cytokine receptor; b. a third transmembrane domain; and c. a second intracellular domain comprising an intracellular domain from the first cytokine receptor. The composition of any one of embodiments 46-57, wherein the third cytokine receptor is IL-2RG (CD132). The composition of any one of embodiments 46-57, wherein the first cytokine receptor is IL-2RG (CD132). The composition of any one of embodiments 46-59, wherein the third transmembrane domain interacts with the first transmembrane domain and/or the second transmembrane domain. The composition of any one of embodiments 46-59, wherein the first transmembrane domain, the second transmembrane domain and the third transmembrane domain form a multimer. The composition of any one of embodiments 46-59, wherein the third transmembrane domain interacts with the first transmembrane domain but not the second transmembrane domain. The composition of embodiment 62, wherein the first transmembrane domain and the third transmembrane domain form a multimer. The composition of any one of embodiments 46-59, wherein the third transmembrane domain interacts with the second transmembrane domain but not the first transmembrane domain. The composition of embodiment 64, wherein the second transmembrane domain and the third transmembrane domain form a multimer. The composition of any one of embodiments 46-65, wherein the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide in a cell that expresses the first, second and third polypeptide compared to a cell that expresses the first polypeptide, the second polypeptide, the third polypeptide, the first and second polypeptide, the first and third polypeptide, the second and third polypeptide or none of the first, second and third polypeptides. The composition of any one of embodiments 46-65, wherein the cell that expresses the first, second and third polypeptides expands at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that expresses only the first polypeptide, only the second polypeptide, only the third polypeptide, the first and second polypeptide, the first and third polypeptide, the second and third polypeptide or none of the first, second and third polypeptides. The composition of embodiment 51, wherein the recombinant polynucleic acid further comprises a fourth sequence encoding a fourth polypeptide comprising a fourth portion of the split receptor switch polypeptide. The composition of embodiment 68, wherein the fourth polypeptide comprises: a. a second extracellular domain from the third cytokine receptor; and b. a fourth transmembrane domain. The composition of embodiment 54, wherein the recombinant polynucleic acid further comprises a fourth sequence encoding a fourth polypeptide comprising a fourth portion of the split receptor switch polypeptide. The composition of embodiment 70, wherein the fourth polypeptide comprises: a. a fourth transmembrane domain, and b. a second intracellular domain comprising an intracellular domain from the third cytokine receptor. The composition of any one of embodiments 68-71, wherein the first intracellular signaling domain is activated at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide in a cell that expresses the first, second, third and fourth polypeptide compared to a cell that expresses the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the first and second polypeptide, the first and third polypeptide, the first and fourth polypeptide, the second and third polypeptide, the second and fourth polypeptide, the third and fourth polypeptide, the first and the second and the third polypeptide, the first and the second and the fourth polypeptide, the first and the third and the fourth polypeptide, the second and the third polypeptide and the fourth polypeptide, or none of the first, second, third and fourth polypeptides. The composition of any one of embodiments 68-71, wherein the cell expressing the first, second, third and fourth polypeptides expands at least 2-fold greater in the presence of a cytokine that binds to the first extracellular domain of the first polypeptide compared to a cell that expresses the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the first and second polypeptide, the first and third polypeptide, the first and fourth polypeptide, the second and third polypeptide, the second and fourth polypeptide, the third and fourth polypeptide, the first and the second and the third polypeptide, the first and the second and the fourth polypeptide, the first and the third and the fourth polypeptide, the second and the third polypeptide and the fourth polypeptide, or none of the first, second, third and fourth polypeptides. The composition of any one of embodiments 1-73, wherein a cell that does not express one or more or each of the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide does not expand in the presence of the cytokine that binds to the extracellular domain of the cytokine receptor switch polypeptide. The composition of any one of embodiments 1-74, wherein the cytokine is IL21. The composition of any one of embodiments 1-75, wherein a. a cell that expresses the first and second polypeptide expands in the presence of IL2 and a cell that does not express the first polypeptide, the second polypeptide, or the first and second polypeptide expands in the presence of IL2; b. a cell that expresses the first and second and third polypeptide expands in the presence of IL2 and a cell that does not express the first polypeptide, the second polypeptide, the third polypeptide, the first and second polypeptide, the first and third polypeptide, the second and third polypeptide or the first and the second and the third polypeptide expands in the presence of IL2; or c. a cell that expresses the first and second and third and fourth polypeptide expands in the presence of IL2 and a cell that does not express the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide, the first and second polypeptide, the first and third polypeptide, the first and fourth polypeptide, the second and third polypeptide, the second and fourth polypeptide, the third and fourth polypeptide, the first and the second and the third polypeptide, the first and the second and the fourth polypeptide, the first and the third and the fourth polypeptide, the second and the third polypeptide and the fourth polypeptide, or the first and the second and the third polypeptide and the fourth polypeptide expands in the presence of IL2. The composition of any one of embodiments 1-76, wherein the first transmembrane domain, the second transmembrane domain, the third transmembrane domain, and/or the fourth transmembrane domain is not from IL21R. The composition of any one of embodiments 1-76, wherein the first transmembrane domain, the second transmembrane domain, the third transmembrane domain, and/or the fourth transmembrane domain is from CD8a, CD8b, CD4, CD28, glycophorm A, EPOR, CD79, DAP10, DAP12, IL21R or a modified IL21R, DAP12, TREM1, KIR2DS2, IgM, CD79a, CD79b, FGFR1, CD3 epsilon, CD3 delta, CD3 gamma, R0R1, LNGFR, IL2RG, or PDGFRa. The composition of any one of embodiments 1-78, wherein the first extracellular domain is from IL21R and the second extracellular domain is from IL-2RG (CD132). The composition of any one of embodiments 1-79, wherein the first intracellular domain comprises an intracellular signaling domain from IL2RB, IL7R, or IL2RG. The composition of any one of embodiments 1-80, wherein the extracellular domain of the cytokine receptor switch polypeptide comprises a sequence with at least about 80% sequence identity to any one of SEQ ID NOs: 54-61. The composition of any one of embodiments 1-81, wherein the transmembrane domain of the cytokine receptor switch polypeptide comprises a sequence with at least about 80% sequence identity to any one of SEQ ID NOs: 62-84. The composition of any one of embodiments 1-82, wherein the intracellular domain of the cytokine receptor switch polypeptide comprises a sequence with at least about 80% sequence identity to any one of SEQ ID NOs: 85-88. The composition of any one of embodiments 1-83, wherein the cytokine receptor switch polypeptide comprises a sequence with at least about 80% sequence identity to any one of SEQ ID NOs: 1-53. The composition of any one of embodiments 1-84, wherein the recombinant polynucleic acid comprises a sequence encoding a chimeric antigen receptor (CAR). The composition of any one of embodiments 36-85, wherein the first protein of interest is a first CAR, the second protein of interest is a second CAR, and the third protein of interest is a third CAR. The composition of embodiment 85 or 86, wherein the recombinant polynucleic acid comprises, in an order from 5’ end to 3’ end, the sequence encoding the CAR and the sequence encoding the first or second polypeptide. The composition of any one of embodiments 85-87, wherein the CAR comprises

(a) an extracellular domain comprising an antigen binding domain;

(b) a transmembrane domain; and

(c) an intracellular domain comprising an intracellular signaling domain. The composition of embodiment 88, wherein the antigen binding domain is an anti-CD19 binding domain. The composition of embodiment 89, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of RASQDISKYLN, SRLHSGV and GNTLPYTFG, respectively; and a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of DYGVS, VIWGSETTYYNSALKS and YAMDYWG, respectively. The composition of embodiment 90, wherein the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID NO: 106 or 107. The composition of embodiment 90, wherein the antigen binding domain comprises an scFv with at least about 80% sequence identity to any one of SEQ ID NOs: 120-143. The composition of embodiment 88, wherein the antigen binding domain is an anti-CD22 binding domain. The composition of embodiment 93, wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively. The composition of embodiment 94, wherein the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID NO: 108. The composition of embodiment 88, wherein the antigen binding domain is an anti-CD20 binding domain. The composition of embodiment 96, wherein the antigen binding domain comprises an scFv with at least about 80% sequence identity to SEQ ID Nos: 144-180. The composition of embodiment 88, wherein the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF -I receptor, GD2, GD3, B7-H3, GPC2, L1CAM, EGFR, mesothehn, MART-1, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE- 1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, CEA, p53, Ras, HER-2, BCR- ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b-Catemn, CDK4, Mum-1, pl 5, pl6, 43-9F, 5T4, 791Tgp72, a-fetoprotein, b-HCG, BCA225, BTAA, CA125, BCAA, CA195, CA242, CA-50, CAM43, CD68/P1, CO-029, FGF-5, G250, Ga733/EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD 19, CD20, CD22, ROR1, CD2 and GD2. The composition of any one of embodiments 88-98, wherein the intracellular domain of the CAR comprises an intracellular signaling domain from CD2. The composition of any one of embodiments 88-98, wherein the intracellular domain of the CAR comprises an intracellular signaling domain from CD3zeta, 4-1BB (CD137), CD28, ICOS, FcyRI, FcRy, FcR, CD3y, CD38, CD3s, CD35, CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70. The composition of any one of embodiments 88-100, wherein the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28. The composition of any one of embodiments 88-101, wherein the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28. The composition of any one of embodiments 88-102, wherein the CAR comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 109-111. The composition of any one of embodiments 88-102, wherein the CAR comprises a sequence with at least 80% sequence identity of any one of SEQ ID Nos: 181-270. The composition of any one of embodiments 13-103, wherein the cell is a lymphocyte. The composition of embodiment 105, wherein the cell is a T cell. The composition of any one of embodiments 13-106, wherein the cell is a population of cells. The composition of embodiment 107, wherein the population of cells comprises at least 1x10 ^A 5 cells. The composition of embodiment 107, wherein at least 60% or more of the cells in the population of cells express at least two CARs. The composition of embodiment 107, wherein at least 60% or more of the cells in the population of cells express at least three CARs. A pharmaceutical composition comprising the composition of any one of embodiments 1-108, and a pharmaceutically acceptable excipient or carrier. A method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of embodiment 111. The method of embodiment 112, wherein the cancer is lymphoma or leukemia. The method of embodiment 112, wherein the method further comprises administering to the subject the cytokine that binds to the cytokine binding domain from a first cytokine receptor. The method of embodiment 112, wherein the method further comprises administering to the subject a cytokine that binds to the extracellular domain of the cytokine receptor switch polypeptide. The method of embodiment 112, wherein the method further comprises administering to the subject IL21 The method of embodiment 112, wherein the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and/or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC). A method of expanding cells, the method comprising culturing the cell of any one of embodiments 13-108 in the presence of the cytokine that binds to the extracellular domain of the cytokine receptor switch polypeptide, thereby expanding the cell. The method of embodiment 118, wherein the cell is expanded at least 3 -fold. A method of making a population of chimeric antigen receptor (CAR)-expressing immune effector cells, comprising

(a) contacting the cell of any one of embodiments 13-108 with a cytokine that binds to the cytokine binding domain from the first cytokine receptor; and

(b) expanding the cell in the presence of the cytokine that binds to the cytokine binding domain from the first cytokine receptor, thereby making a population of chimeric antigen receptor (CAR)- expressing immune effector cells. A method of modulating activity of a chimeric antigen receptor (CAR)-expressing immune effector cell, comprising contacting the cell of any one of embodiments 13-108 with a cytokine that binds to the cytokine binding domain from the first cytokine receptor, wherein the cell comprises a CAR, thereby modulating activity of a chimeric antigen receptor (CAR)-expressing immune effector cell. The method of embodiment 121 wherein activity of a cell not expressing a CAR is modulated to a lesser extent by contacting the cell with a cytokine that binds to the cytokine binding domain from the first cytokine receptor than the extent of modulation of activity of a cell that comprises the CAR that is contacted with the cytokine.

EXAMPLES

[0227] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature cited herein.

[0228] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

Example 1 - Exemplary split receptor polypeptide constructs

[0229] Exemplary split receptor polypeptide constructs are listed in Table 1.

Table 1: Exemplary split receptor polypeptide constructs

[0230] “R” refers to receptor, for example, “IL21R” refers to IL21 receptor; “IL2RB” refers to IL2 receptor beta; “IL7R” refers to IL7 receptor; “IL2R” refers to IL2 receptor; “IL2RG” refers to IL2 receptor common gamma chain; “IL4R” refers to IL4 receptor; “IL9R” refers to IL9 receptor; “IL15R” refers to IL15 receptor; “IL2Ra” refers to IL2 receptor alpha; “LNGFR” refers to low affinity nerve growth receptor; “PDGFRa” refers to platelet derived growth factor receptor alpha; “Ecto” refers to extracellular domain. “TM” refers to transmembrane domain. “Endo” refers to intracellular domain.

Example 2 - Exemplary CAR constructs

[0231] Exemplary antigen binding domains for the CAR are listed in Table 2.

Table 2: Exemplary Antigen Binding Domains for CAR

[0232] Exemplary CAR constructs are listed in Table 3.

Table 3: Exemplary CAR Constructs

Example 3 - Selective Expansion of Transduced Cells

Synthesis of split receptor switch polypeptides

[0233] Codon optimized genes encoding the split receptor switch polypeptides were synthesized and cloned into retrovirus vector MSGV 1. Viral supernatant was prepared by transfecting HEK293gp with MSGV1 using lipofectamine. After 2 days, supernatant containing virus was harvested, filtered and centrifuged for 2 hours at 3000g onto retronectin coated 24 well plates.

T-cell transduction

[0234] T cells were activated on day 0 using Transact (Miltinyi Biotech). On day 2, T cells were transferred to 24-well retronectin plates containing retrovirus for transduction. T cells were grown with either IL-2 for general expansion of all cells or with IL-21 for specific expansion of cytokine receptor cells. On day 6, T cells were transferred to GRex plates for the remainder of cell expansion. Cytokines were added every 2-3 days during the experiment, and cells were counted to determine cell proliferation. As shown in FIGs. 3B and 4, IL2 promoted expansion of both transduced and nontransduced cells; however, IL21 could promote the expansion of only the transduced cells but not the non-transduced cells.

[0235] Primary human PBMCs were isolated from leukoreduction system (LRS) chambers using a Ficoll gradient. T cells were isolated with magnetic beads using a Pan T cell isolation kit (Miltenyi). Isolated T cells were frozen and stored in liquid nitrogen until being used for viral transduction. Example 4 - Optimization of Split IL21/LL2RB Receptor

[0236] A set of split receptors containing the IL21R extracellular domain on the first polypeptide and the IL2RB intracellular domain on the second polypeptide were designed with various dimerizing transmembrane domains to assess receptor function. In experiment 1, the IL21R/IL2RB split receptor switch polypeptide was used as a positive control for growth in media containing IL-21.

[0237] In a second experiment, fluorescent proteins mNeonGreen and mtagBFP2 were co-expressed with the split receptors to assess transduction efficiency and track the selection of double positive cells. Transduced cells were grown in the presence of IL-2 as a control to support growth of all cells or IL- 21 to support growth of cells with an intact, functional IL21R/IL2RB split receptor switch polypeptide. TMs tried were DAP12 homodimers with or without associated TREM1 or KIR2DS2 TM proteins, CD79a and CD79b heterodimers with or without associated membrane IgM, glycophorin A TM, CD28 TM and FGFR1 TM. The results of experiment 2 are shown in FIG. 3 A, FIG. 3B and FIG. 4.

[0238] The split receptors based upon DAP 12 TMs (SEQ ID NO: 46+SEQ ID NO: 47, SEQ ID NO: 46+SEQ ID NO: 49, SEQ ID NO: 47+SEQ ID NO: 48, SEQ ID NO: 42+SEQ ID NO: 43) supported cell growth and double positive cell selection with IL-21 treatment. The split receptor that supported the best growth had IL21R on a TREM1 TM and IL2RB on a DAP 12 TM (SEQ ID NO: 47 + SEQ ID NO: 48). The flow cytometry plots show that this split receptor can enrich for cells that are double positive for the co-expressed fluorescent proteins (FIG. 4).

Example 5 - Testing Various TMs in Swapped IL21R/IL2RB Split Receptors

[0239] A set of swapped receptors containing the IL21R extracellular domain paired with IL2RG intracellular signaling domain on one protein and the IL2RG extracellular domain paired with IL2RB intracellular signaling domain on a second protein were designed with different transmembrane domains (TMs) to assess which TMs could combine to reconstitute receptor function (FIG. 5A, FIG. 5B, and FIG. 5C). Transduced cells were grown in the presence of IL-2 as a control to support growth of all cells or IL-21 to support growth of cells with an intact, functional split receptors. TMs tried were ROR1, LNGFR, IL2RG, IL21R and PDGFRa. Swapped proteins containing nonfunctional truncated IL2RB and IL2RG intracellular signaling domains were used as negative controls. Split receptors containing IL21R/IL2RB (SEQ ID NO: 2 and SEQ ID NO: 3) were used as positive controls for IL-21 driven cell growth and full length IL21R (SEQ ID NO: 1) was used as a negative control for IL-21 signaling but no cell growth. The results are shown in FIG. 6.

[0240] All tested TMs were able to support IL-21 driven cell growth using the swapped receptors when both receptors were co-expressed. None of the receptors containing truncated signaling domains or when transduced as a single receptor were able to support cell growth with IL-21 treatment. Example 6 - Selection of multi-edited T cells using Split Receptor Switch Polypeptides

[0241] In this experiment T cells were transduced with a first vector containing a sequence encoding a first portion of a split receptor that has an IL-21R extracellular domain with a TREM1 TM, a sequence encoding a CD 19 CAR, and a sequence encoding a CD20 CAR (SEQ ID NO: 119), and a second vector containing a sequence encoding a second portion of the split receptor that has an IL2RB intracellular domain with a DAP12 TM, and a sequence encoding a CD22 CAR (SEQ ID NO: 118). The transduced cells were grown in media without cytokine added, with IL-21 added or with IL-2 added. On day 10, T cells with both vectors integrated expanded preferentially in IL-21 containing media compared to media without cytokines added or with IL-2 added. As shown in FIG. 7, 65.61% of T cells were CD19CAR+CD22CAR+ in the media with IL-21 added, whereas only 25.41% of T cells were CD19CAR+CD22CAR+ in the media with no cytokine added and only 21.57% of T cells were CD19CAR+CD22CAR+ in the media with IL-2 added. The result demonstrates that using the methods described herein a purer multi-edited T cell population can be obtained compared to conventional methods. For example, using the methods described herein a higher percentage of the T cells that express both a first CAR encoded by a first vector (CD 19 CAR), and a second CAR encoded by a second vector (CD22 CAR) can be obtained compared to conventional methods. As demonstrated in this example, transduction of a population of T cells with a first vector having a sequence encoding the first CAR and a sequence encoding a first portion of a split receptor switch polypeptide, and a second vector having a sequence encoding the second CAR and a sequence encoding a second portion of a split receptor switch polypeptide followed by incubation of the cells in the presence of a cytokine that binds to the first portion of a split receptor switch polypeptide produced a population of T cells in which a higher percentage of the T cells in the population expressed both the first CAR and the second CAR compared to the percentage of the T cells that expressed both the first CAR and the second CAR when the T cell population was transduced with a first vector encoding the first CAR but without the sequence encoding the first portion of the split receptor switch polypeptide, and a second vector encoding the second CAR but without the sequence encoding the second portion of the split receptor switch polypeptide.

[0242] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.